RESUMO
Hepatocyte cell adhesion molecule (hepaCAM) encodes a generally inactive phosphorylated glycoprotein which mediates cancer cell proliferation, migration, and differentiation. We have reported that hepaCAM is down-regulated in renal cell carcinoma (RCC) and takes responsibility of cell growth inhibition. However, the precise mechanisms of hepaCAM inhibits cell growth is still unknown. In this study, we demonstrated that re-expression of hepaCAM can cause an accumulation in G0/G1 phase in 786-0 cells. This reaction was accompanied by a substantial reduction of c-Myc expression through using an ectopic hepaCAM expression system. Furthermore, we found a comparable decrease in proliferation and G0/G1 accumulation of 786-0 and RC-2 cells after treatment with a small molecule c-Myc inhibitor, 10058-F4. This indicated that the down regulation of c-Myc was an essential process in controlling growth inhibitory actions of hepaCAM. Nevertheless, re-expression of hepaCAM results in apparent reduction of c-Myc protein with no corresponding reduction of c-Myc mRNA. This suggests that this reaction might take place at a post-transcriptional level rather than transcriptional one. Consistent with these findings, hepaCAM decreased c-Myc stability by increasing the proportion of c-Myc phosphorylation on T58 which can be abrogated by a proteasomal inhibitor (MG132). Thus, our research implies that the decrease in c-Myc protein expression, resulting from ectopic expression of hepaCAM, may contribute to the inhibition of proliferation in these cells.
Assuntos
Carcinoma de Células Renais/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Fase G1 , Humanos , Técnicas In Vitro , Estabilidade Proteica/efeitos dos fármacos , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologiaRESUMO
Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatocytes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/química , Animais , Células Cultivadas , Hepatócitos/citologia , Ensaios de Triagem em Larga Escala/instrumentação , Masculino , Ratos , Ratos WistarRESUMO
AIM: We aimed to investigate the mechanisms of hepaCAM inactivation in transitional cell carcinoma of the bladder through the analysis of hepaCAM exon 2 methylation. METHODS: The methylation of hepaCAM exon 2 and the expression of hepaCAM were determined by methylation-specific restriction PCR assay and RT-PCR in bladder cancer cells (T24, BIU-87) as well as in 55 paired bladder cancer specimens. The methylated bladder cancer cells were treated with 5-Aza- 2'-deoxycytidine (5-Aza-CdR), a demethylating agent. MTT was used to detect the proliferation of T24 and BIU-87 cells. RESULTS: The proliferation of T24 and BIU-87 cells was suppressed by treatment with different concentrations of 5-Aza-CdR; the expression of hepaCAM was absent in T24 and BIU-87 cells, and we found that exon 2 of hepaCAM was methylated in the 2 cells. hepaCAM mRNA was re-expressed and the methylation status of hepaCAM exon 2 was reversed after treatment with 5-Aza-CdR. The expression of hepaCAM mRNA in bladder cancer tissues was significantly lower than that in adjacent tissues. The methylation rate of hepaCAM exon 2 was significantly higher in bladder cancer tissues than in adjacent tissues. The methylation of hepaCAM exon 2 was related to hepaCAM expression in bladder cancer tissues. CONCLUSIONS: Downregulation of hepaCAM expression plays an important role in the tumorigenesis and development of bladder cancer. DNA methylation may be important for downregulation of hepaCAM expression in bladder cancer.
Assuntos
Carcinoma de Células de Transição/genética , Metilação de DNA , Éxons , Proteínas/metabolismo , Neoplasias da Bexiga Urinária/genética , Idoso , Azacitidina/farmacologia , Carcinoma de Células de Transição/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.
Assuntos
Dendrímeros/química , Engenharia Tecidual/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Dendrímeros/efeitos adversos , Humanos , Microscopia Eletrônica de Varredura , Poliaminas/química , Polietilenoglicóis/químicaRESUMO
We previously reported the identification and characteristics of hepaCAM, a new immunoglobulin-like adhesion molecule. Frequently lost in diverse tumors, hepaCAM exhibits antiproliferative effects on cancer cells and promotes cell-extracellular matrix (ECM) interactions when re-expressed. Herein, we demonstrate for the first time that hepaCAM is cleaved in the human breast carcinoma MCF7 cells, generating a fragment containing mainly the cytoplasmic domain. The phorbol ester phorbol 12-myristate 13-acetate (PMA) did not affect the cleavage of hepaCAM. However, calcium-influx promoted hepaCAM cleavage independent of PKC. In addition, inhibitors of proteasome and cysteine proteases strongly suppressed the cleavage of hepaCAM, indicating the involvement of proteasome, calpain-1 and cathepsin B. Furthermore, we showed that functions of hepaCAM were impaired when the cytoplasmic domain was cleaved. The truncation mutant of hepaCAM failed to promote cell-ECM adhesion and migration, and lost the inhibitory effects on cell growth, suggesting a regulatory role of the cleavage in hepaCAM functions.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Neoplasias da Mama/patologia , Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Carcinoma/patologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Humanos , Imunoglobulinas/química , Modelos Biológicos , Peso Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína , Proteínas/química , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
For acute, chronic, or hereditary diseases of the liver, cell transplantation therapies can stimulate liver regeneration or serve as a bridge until liver transplantation can be performed. Recently, fetal hepatocytes, stem cells, liver progenitor cells, or other primitive and proliferative cell types have been employed for cell transplantation therapies, in an effort to improve the survival, proliferation, and engraftment of the transplanted cells. Reviewing earlier studies, which achieved success by transplanting mature hepatocytes, we propose that there is a switch-like regulation of liver regeneration that changes state according to a stimulus threshold of extracellular influences such as cytokines, matrices and neighboring cells. Important determinants of a successful clinical outcome include sufficient quantities and functional levels of the transplanted cells (even for short periods to alter the environment), rather than just engraftment levels or survival durations of the exogenously transplanted cells. The relative importance of these determining factors will impact future choices of cell sources, delivery vehicles, and sites of cell transplantation to stimulate liver regeneration for patients with severe liver diseases.
Assuntos
Transplante de Células/métodos , Hepatopatias/cirurgia , Regeneração Hepática , Animais , Proliferação de Células , Sobrevivência Celular , Hepatócitos/transplante , Humanos , Hepatopatias/patologia , Índice de Gravidade de Doença , Alicerces Teciduais , Resultado do TratamentoRESUMO
OBJECTIVES: To evaluate hepaCAM (hepatocyte cell adhesion molecule) gene expression in patients with renal cell carcinoma (RCC) and to explore its effect on proliferation of 786-0 cells. hepaCAM is a tumor suppressor gene, which has been identified as a member of immunoglobulin superfamily cell adhesion molecule. METHODS: Two-step reverse transcription-polymerase chain reaction was used to determine hepaCAM expression in 30 paired (RCC and the adjacent non-RCC) renal specimens. Transfection studies were carried out by expressing green fluorescent protein and green fluorescent protein-fused hepaCAM in 786-0 cells. RESULTS: Significant downregulation of hepaCAM was detected in 25 of 30 RCC patients tested. When transfected into 786-0 cells, the number of colony formation was reduced by 5-fold according to colony formation assay. MTT (3-diphenyltetrazolium bromide) showed the inhibition rates on the fourth, fifth, and sixth days of culturing were 26.5%, 38.1%, and 35.7%, respectively. CONCLUSION: Our data show that hepaCAM is frequently downregulated in RCC, and that exogenous hepaCAM exhibits antiproliferative effect on 786-0 cells, suggesting that silencing of hepaCAM may be associated with carcinogenesis of RCC.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas/genética , Adulto , Idoso , Proteínas de Ciclo Celular , Proliferação de Células , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas/fisiologiaRESUMO
In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.
Assuntos
Moléculas de Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Humanos , Invasividade NeoplásicaRESUMO
Subsequent to our identification of a novel immunoglobulin-like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re-expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373-MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373-MG cell lines expressing hepaCAM at different levels. The results revealed that high-level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low-level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373-MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM.
Assuntos
Moléculas de Adesão Celular/fisiologia , Diferenciação Celular , Glioblastoma/patologia , Proteínas/fisiologia , Adesão Celular , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Humanos , Proteínas/análise , Proteínas/genéticaRESUMO
Previously, we reported the identification of a novel immunoglobulin-like cell adhesion molecule hepaCAM that promotes cell-extracellular matrix (ECM) interactions including cell adhesion and motility. Cell-ECM interactions are known to be directed by the actin cytoskeleton. In this study, we examined the association of hepaCAM with the actin cytoskeleton. We found that hepaCAM was partially insoluble in Triton X-100 and colocalized with the actin cytoskeleton on the plasma membrane. Disruption of F-actin decreased the detergent insolubility and disturbed the subcellular localization of hepaCAM. Coimmunoprecipitation and F-actin cosedimentation assays revealed that hepaCAM directly bound to F-actin. In addition, we constructed three N- and C-terminal domain-deleted mutants of hepaCAM to determine the actin-binding region as well as to evaluate the effect of the domains on the biological function of hepaCAM. Detergent solubility assays showed that the cytoplasmic domain of hepaCAM might be required for actin association. However, deletion of either the extracellular or the cytoplasmic domain of hepaCAM abolished actin coprecipitation as well as delayed cell-ECM adhesion and cell motility. The data suggest that an intact hepaCAM protein is critical for establishing a stable physical association with the actin cytoskeleton; and such association is important for modulating hepaCAM-mediated cell adhesion and motility.
Assuntos
Actinas/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Proteínas/metabolismo , Actinas/genética , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Detergentes/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Proteínas/genéticaRESUMO
Subsequent to our identification of the novel immunoglobulin-like cell adhesion molecule hepaCAM, we demonstrated that hepaCAM is capable of modulating cell growth and cell-extracellular matrix interactions. In this study, we examined the localization of hepaCAM in lipid rafts/caveolae as well as the interaction of hepaCAM with the caveolar structural protein caveolin-1 (Cav-1). Our results revealed that a portion of hepaCAM resided in detergent-resistant membranes and co-partitioned with Cav-1 to low buoyant density fractions characteristic of lipid rafts/caveolae. In addition, co-localization and coimmunoprecipitation assays confirmed the association of hepaCAM with Cav-1. Deletion analysis of hepaCAM showed that the extracellular first immunoglobulin domain of hepaCAM was required for binding Cav-1. Furthermore, when co-expressed, Cav-1 induced the expression of hepaCAM as well as distributed hepaCAM to intracellular Cav-1-positive caveolar structures. Taken together, our findings indicate that hepaCAM is partially localized in the lipid rafts/caveolae and interacts with Cav-1 through its first immunoglobulin domain.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Imunoglobulinas/química , Camundongos , Células NIH 3T3 , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas/genética , Deleção de Sequência , Transdução de SinaisRESUMO
Previously, we reported the identification of a novel immunoglobulin-like cell adhesion molecule hepaCAM that is frequently downregulated and inhibits cell growth in hepatocellular carcinoma. In this study, we show that the expression of hepaCAM is suppressed in diverse human cancers. Aiming to evaluate the biological role of hepaCAM in breast cancer, we stably transfected the MCF7 cell line with either wild-type hepaCAM or its mutant hCAM-tailless that lacked the cytoplasmic domain. We found that hepaCAM inhibited colony formation and cell proliferation and arrested cells in the G(2)/M phase. Intriguingly, hepaCAM was capable of inducing cellular senescence as defined by the enlarged cell morphology and increased beta-galactosidase activity. Furthermore, hepaCAM elevated the expression levels of senescence-associated proteins including p53, p21 and p27. In contrast, cell growth inhibition and senescence were less apparent in cells overexpressing hCAM-tailless mutant. To determine if the p53-mediated pathway was involved in hepaCAM-induced senescence, we used the small-interfering RNA system to knock down endogenous p53 expression. In the presence of hepaCAM, downregulation of p53 resulted in a clear reduction of p21, insignificant change in p27 and alleviated senescence. Together, the results suggest that the expression of hepaCAM in MCF7 cells not only inhibits cell growth but also induces cellular senescence through the p53/21 pathway.
Assuntos
Neoplasias da Mama/metabolismo , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Neoplasias da Mama/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo , Feminino , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
Genetic alterations have been defined as the hallmark of cancers as they are responsible for the differences between normal and malignant phenotypes. A widely accepted approach to study genetic instability is to identify cancer-related genes, in particular, the two major groups of growth regulatory genes - oncogenes and tumour suppressor genes. Using the technique of suppression subtractive hybridisation, we identified a novel gene transcript, designated as HEPT3. RT-PCR demonstrated that HEPT3 was overexpressed in 87% (20/23) of HCC patients and in 4/5 HCC cell lines tested. Sequence analyses performed on the full-length cDNA revealed that HEPT3 is an intronless gene mapped to human chromosome 6q13-14. The gene transcript lacks an extensive open reading frame and contains an Alu sequence near the 5' terminus, indicating that HEPT3 encodes a noncoding RNA. Antisense studies on the HCC cell line HepG2 showed that, when HEPT3 expression level was reduced, cell proliferation rate was inhibited by approximately 5-fold and cell colony formation was reduced by at least 50%. Our data suggest that the novel gene HEPT3 may function through its noncoding RNA and its overexpression may play a role in hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/biossíntese , Sequência de Bases , Linhagem Celular Tumoral , Cromossomos Humanos Par 6 , Primers do DNA/química , DNA Complementar/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos Antissenso/química , RNA Neoplásico/metabolismo , RNA não Traduzido , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: Previously, we reported on gene HEPN1 that was silenced in hepatocellular carcinoma (HCC) and its capability of arresting cell growth. In this study, we identified another novel gene hepaCAM from the liver, which contains the full-length HEPN1 on its antisense strand in the 3'-noncoding region, and assessed its expression, characteristics and functions in HCC. METHODS: Full-length hepaCAM cDNA was isolated by rapid amplification of cDNA ends. The gene expression was examined by semi-quantitative RT-PCR in 23 paired HCC liver specimens and 5 HCC cell lines. Transfection studies, coupled with immunocytochemistry, cellular interaction analyses, colony formation and microtetrazolium assay, were employed to elucidate the localization and functions of hepaCAM. RESULTS: The expression of hepaCAM decreased in 20/23 of HCC samples and was undetectable in 5 HCC cell lines tested. The gene product consisting of 416 amino acids displayed the typical structure of Ig-like cell adhesion molecules. The protein was glycosylated and predominantly localized on the cytoplasmic membrane. When re-expressed in HepG2, hepaCAM accelerated cell spreading (P<0.001), increased cell motility (P=0.0011), reduced colony formation (P=0.0022), and inhibited cell growth (P<0.001). CONCLUSIONS: Gene hepaCAM, frequently silenced in HCC, encodes an Ig-like transmembrane glycoprotein and is involved in cell adhesion and growth control.
Assuntos
Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/genética , Neoplasias Hepáticas/genética , Proteínas/genética , Sequência de Aminoácidos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Clonagem Molecular , Glicosilação , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
We have recently identified a novel gene, hepaCAM, in liver that encodes a cell adhesion molecule of the immunoglobulin superfamily. In this study, we examined the characteristics of hepaCAM protein and the relationship between its structure and function, in particular its adhesive properties. The wild-type and the cytoplasmic domain-truncated mutants of hepaCAM were transfected into the human breast carcinoma MCF7 cells, and the physiological and biological properties were assessed. Biochemical analyses revealed that hepaCAM is an N-linked glycoprotein phosphorylated in the cytoplasmic domain and that it forms homodimers through cis-interaction on the cell surface. The subcellular localization of hepaCAM appears density-dependent; in well spread cells, hepaCAM is distributed to cell protrusions, whereas in confluent cells, hepaCAM is predominantly accumulated at the sites of cell-cell contacts on the cell membrane. In polarized cells, hepaCAM is recruited to the lateral and basal membranes, and lacking physical interaction, hepaCAM is shown to co-localize with E-cadherin at the lateral membrane. Cell adhesion and motility assays demonstrated that hepaCAM increased cell spreading on the matrices fibronectin and matrigel, delayed cell detachment, and enhanced wound healing. Furthermore, when the cytoplasmic domain was deleted, hepaCAM mutants did not affect cell surface localization and dimer formation. Cell-matrix adhesion, however, was less significantly increased, and cell motility was almost unchanged when compared with the effect of the wild-type hepaCAM. Taken together, the cytoplasmic domain of hepaCAM is essential to its function on cell-matrix interaction and cell motility.
Assuntos
Neoplasias da Mama/patologia , Proteínas/metabolismo , Neoplasias da Mama/química , Caderinas/metabolismo , Adesão Celular , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Polaridade Celular , Extensões da Superfície Celular/química , Dimerização , Feminino , Glicoproteínas , Humanos , Junções Intercelulares/química , Fosfoproteínas , Transporte Proteico , Proteínas/química , Proteínas/fisiologia , TransfecçãoRESUMO
BACKGROUND/AIMS: Examining genes associated with human hepatocellular carcinoma (HCC) by subtractive hybridisation, we identified a novel transcript, designated as HEPN1, in non-tumorous liver. In this study, we aimed to evaluate HEPN1 gene expression in HCC patients, to characterise and to explore the functional significance of HEPN1 in vitro. METHODS: One-step reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were employed to determine HEPN1 expression in 23 paired (HCC and the adjacent non-HCC) liver specimens. Sequence analyses were performed by bioinformatics. Transfection studies were carried out by expressing HEPN1, V5-fused HEPN1, and green fluorescent protein-fused HEPN1, individually, in HepG2 cells. RESULTS: Significant downregulation of HEPN1 (P<0.0001) was detected in 22/23 of HCC patients tested. Gene HEPN1 maps to chromosome 11q24.2; and the predicted gene product, a 10-kDa peptide with 88 amino acids, has no homology to known proteins. When transfected into HepG2 cells, HEPN1 reduced cell viability to 37.5+/-2.5% (P=0.001), and induced apoptosis with typical morphological changes as demonstrated by microscopy and Annexin V assay. CONCLUSIONS: Our data show that HEPN1 is frequently silenced in HCC, and that exogenous HEPN1 exhibits antiproliferative effect on HepG2 cells, suggesting that silencing of HEPN1 may be associated with carcinogenesis of hepatocytes.