RAI , one candidate gene associated with differentiation of human lung adenocarcinoma cells

From all-trans retinoic acid (ATRA)-treated human lung adenocarcinoma GLC-82 cells and control, subtractive cDNA library has been constructed using subtractive hybridization technique in our laboratory. The screening of the cDNA subtractive library resulted in identification of a clone containing cDNA fragment of one ATRA-induced gene (RAI) in GLC-82 cells. The positive clone with full-length cDNA of RAI was identified by screening fetal brain cDNA library using colony hybridization technique, and then sequenced. RT-PCR results showed that RAI was expressed in many different human fetal tissues. These results suggest that RAI may be involved in cell differentiation and play an important role in vital activities of cells.     

A case of resectable lung adenocarcinoma associated with sarcoidosis

A 71-year-old woman with uveitis was referred to our hospital for further examination of the possible underlying diseases. In roentgenological examination with plain X-ray and CT scan, hilar and mediastinal lymphadenopathy and a mass shadow in the right upper lung field was observed, whereas fibrotic changes were not obvious in both lung fields. Transbronchial lung biopsy with fiberoptic bronchoscope revealed granulomatous interstitial pneumonia. CD4-positive lymphocytes were increased in bronchoalveolar lavage. The patient was diagnosed as having sarcoidosis. Subsequently, right upper lobectomy was performed, and Stage I lung adenocarcinoma was diagnosed. The patient is under follow up without medication and the disease has been stable for two years. A relationship between epithelioid granulomatosis and malignant diseases is discussed and a review of the literature is given. Since it is still controversial as to the incidence of malignant diseases in sarcoidosis patients, it is important to accumulate data on these associations.     

Chromatin patterns associated with lung adenocarcinoma progression

The development and progression of lung adenocarcinoma, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes and the role of chromatin structure in malignant transformation remains poorly understood. We used systematic nucleosome distribution and chromatin accessibility microarray mapping platforms to analyze the genome-wide chromatin structure from normal tissues and from primary lung adenocarcinoma of different grades and stages. We identified chromatin-based patterns across different patients with lung adenocarcinoma of different cancer grade and stage. Low-grade cancers had nucleosome distributions very different compared with the corresponding normal tissue but had nearly identical chromatin accessibility. Conversely, nucleosome distributions of high-grade cancers showed few differences. Substantial disruptions in chromosomal accessibility were seen in a patient with a high-grade and high-stage tumor. These data imply that chromatin structure changes during the progression of lung adenocarcinoma. We have therefore developed a model in which low-grade lung adenocarcinomas are linked to changes in nucleosome distributions, whereas higher-grade tumors are linked to large-scale chromosomal changes. These results provide a foundation for the development of a comprehensive framework linking the general and locus-specific roles of chromatin structure to lung cancer progression. We propose that this strategy has the potential to identify a new class of chromatin-based diagnostic, prognostic and therapeutic markers in cancer progression.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice     

Retinoic acid regulates,in vitro,the two normal pathways of differentiation of human laryngeal keratinocytes

Summary We have investigated the regulation of the two normal differentiation pathways followed by laryngeal epithelium. Using a tissue culture system that permits growth of cells at the air-liquid interface in serum-free medium, we found that modulating the concentration of retinoic acid is sufficient to determine which pathway is used. At 10⁻⁸ M retinoic acid, the cells form a stratified squamous epithelium which expresses the differentiation-specific keratin K13. At 10⁻⁷ M retinoic acid, the cells form a ciliated pseudostratified epithelium, with no expression of K13. These results are distinct from those seen with foreskin keratinocytes, which have only a single pathway of normal differentiation. This work was supported by grant 3 P01 DC00203 from the National Institute on Deafness and Other Communication Disorders, Bethesda, MD (B.M.S., A.L.A. and T.P.D.) and grants from the Otolaryngology Foundation, Capt. Leo Berger, and the Morris S. and Florence H. Bender Foundation (M.G.M.).     

The role of retinoic acid receptor inhibitor LE135 on the osteochondral differentiation of human bone marrow mesenchymal stem cells

The present study aimed to investigate the role of a retinoic acid receptor-β (RARβ) inhibitor LE135 on TGF-β induced chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs). Pellet culture with exogenous transforming growth factor-β (TGF-β), and a mechanically loaded scaffold system were used to provide two culture models. All samples were cultured for 8 days and changes in early gene expression were determined. Glycosaminoglycan and mRNA expression data showed that LE135 itself did not induce any chondrogenic response in either pellet culture or scaffold culture of hMSCs. LE135 actually inhibited the chondrogenic response caused by exogenous TGF-β, or endogenous TGF-β induced by mechanical load, while the expression of genes normally associated with osteogenesis was not affected. This suggests that the inhibitor LE135 affects the osteochondral differentiation pathway at a different stage, inhibiting chondrogenic gene expression while having no effect on genes normally associated with the osteogenic phenotype. Alternatively, it might be that different cells were proceeding down different lineages. Some cells were undergoing chondrogenesis and this was affected by LE135, while other cells underwent osteogenic differentiation and were not affected by LE135.     

Methylation of CLEC14A is associated with its expression and lung adenocarcinoma progression

Our main objective is probing the effect of methylation of CLEC14A on its expression and lung adenocarcinoma (LUAD) progression. Microarray analysis was utilized to screen out differentially downregulated genes with hypermethylation in LUAD tissues. The CLEC14A expression level was measured by western blot analysis and qRT-PCR. Methylation-specific-PCR was performed to evaluate methylation status of CLEC14A. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid (MTT) assay was used to check the relation between CLEC14A expression and cell proliferation. Cell cycle, cell apoptosis, migration, and invasion were respectively detected by the flow cytometry assay, wound healing assay, and transwell assay. Tumor xenograft models were established for investigating the effect of CLEC14A on tumor formation. CLEC14A expression in LUAD tissues was impaired compared with that in adjacent tissues, and CLEC14A promoter was highly methylated in LUAD. Overexpressing CLEC14A or inhibiting the methylation level of CLEC14A in A549 and LTEP-a-2 cells impeded the duplication of LUAD cells, promoted apoptosis, attenuated cell migration, and invasion ability, and arrested cell cycle at the G0/G1 phase. Overexpression of CLEC14A inhibited tumorigenesis of LUAD cells in nude mice. The promoter of CLEC14A is methylated in LUAD, leading to downregulation of CLEC14A in LUAD. CLEC14A acts as an antitumor role in LUAD by suppressing cell proliferation, migration, invasion, promoting cell apoptosis, and reducing tumorigenicity in nude mice. Thus, the inhibition of CLEC14A methylation is a novel strategy for the clinic treatment of LUAD.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells

Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.     

Establishment and characterization of a nerve cell line (NC-HIMT) from HIMT cells derived from a human ovarian immature teratoma with special reference to the induction of neuron differentiation by retinoic acid

A nerve cell line designated NC-HIMT was established from a HIMT cell line derived from a benign ovarian, three germ layer immature teratoma removed from a 21-year-old Japanese female. The HIMT cells were elongated, ellipsoid or spherical in shape, whose karyotype was on the high side of normal diploidy. Small amounts of retinoic acid enhanced differentiation and maturation of the HIMT cells into nervous tissue, and the NC-HIMT cell line was established by the colony isolating technique when the HIMT cell line was cultured in the presence of retinoic acid-supplemented medium. After establishment, the NC-HIMT cell line was cultured and maintained in retinoic acid-free growth medium. Even though these cells were cultured without retinoic acid, the phenotype of nerve cells remained and the cells were also maintained in a state of high normal diploidy. The nerve cells contacted each other with their long cell projections and formed networks. Immunocytochemical observations using anti-bovine NSE, alpha-internexin, neurofilament 200kD, peripherin and GFAP confirmed that the cells were either nerve cells or glia cells. These results assume that HIMT cells, which were derived from an immature teratoma, have progenitor and/or stem cells which can differentiate into nerve and/or glial cells.     

TRB3 interacts with ERK and JNK and contributes to the proliferation,apoptosis, and migration of lung adenocarcinoma cells

Tribbles homolog 3 (TRB3) has been accounted for regulation of a few cell processes through interaction with other significant proteins. The molecular mechanisms underlying TRB3 in tumorigenesis in lung adenocarcinoma have not been entirely elucidated. The present study is aimed at determining the function and fundamental mechanisms of TRB3 in lung adenocarcinoma progression. TRB3 was highly expressed in A549 and H1299 cells and lung adenocarcinoma tissues compared with human bronchial epithelial cells (HBEpC) and adjacent normal lung tissues. Hypoxia significantly upregulated the expression of TRB3 protein in A549 and H1299 cells in a time-dependent way. Gene expression profiling interactive analysis data analysis indicated that patients with lung adenocarcinoma with excessive expression of TRB3 mRNA had fundamentally shorter survival time. TRB3 knockdown in A549 cells can inhibit cell proliferation and migration, and promote cell apoptosis. TRB3 knockdown reduced the expression of p-ERK and p-JNK, but did not affect the expression of p-P38 MAPK. TRB3 overexpression enhances the malignant transformation abilities of HBEpC such as cell proliferation, migration and colony formation, which could be reversed by U0126 and SP600125. TRB3 overexpression promotes the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but was not affected by U0126 and SP600125. The results of coimmunoprecipitation experiments indicated that TRB3 binds directly to ERK and JNK. This study suggests that TRB3 has a potentially carcinogenic role in lung adenocarcinoma by binding to ERK and JNK and promoting the phosphorylation of ERK and JNK. TRB3 can be a possible therapeutic focus for lung adenocarcinoma.     

3,4-Dihydroxyphenylalanine (DOPA) metabolism and retinoic acid induced differentiation in human neuroblastoma

In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of DOPA. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of DOPA and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-59, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.     

Cloning of human myeloid-associated differentiation marker (MYADM) gene whose expression was up-regulated in NB4 cells induced by all-trans retinoic acid

A full-length cDNA of 3192 bp isolated from human bone marrow cDNA library was predicted an ORF encoding 298 amino acids. The deduced protein, containing seven putative transmembrane segments and sharing 75.8% amino acid identity with mouse Myadm protein, was named as human MYADM. The results of Northern blot analysis showed that MYADM was ubiquitously expressed in 15 of 16 adult tissues tested, except thymus. To determine whether the novel human gene was involved in hematopoietic differentiation process as mouse Myadm did, we examined the mRNA expressive abundance of this gene between normal bone marrow cells and peripheral blood leukocytes, and detected the expression change in NB4 cells induced by all–trans retinoic acid at different induce time by the semi-quantitative RT-PCR. The results showed that the expression of the novel gene was not only significantly higher in peripheral blood leukocytes than in bone marrow cells, but also significantly up-regulated when the NB4 cells(derived from a patient with acute promyelocytic leukemia) were induced by all-trans retinoic acid (ATRA) for 48hr. It is suggested that human MYADM was also associated with the differentiation of hematopoietic cells or acute promyelocytic leukemia cells. In addition, MYADM was mapped to human chromosome 19q13.33-q13.4 by Radiation Hybrid mapping, and it consists of 3 exons and 2 introns and spans a 7.1-Kb genomic region.     

Expression of lipoprotein lipase associated with lung adenocarcinoma tissues

Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.     

Dickkopf‐3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells

Dickkopf‐3 (Dkk‐3) and Dkkl‐1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera‐2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk‐3 but not of Dkkl‐1. Ectopic expression of both Dkk‐3 and Dkkl‐1 induced apoptosis in NT2 cells. Gene silencing of Dkk‐3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk‐3‐silenced cells, this morphological response was not observed in Dkk‐3‐silenced cells. These findings suggest that Dkk‐3 plays a role in the regulation of cell interactions during RA‐induced neuronal differentiation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1243–1254, 2014