A comparative analysis of DNA methylation across human embryonic stem cell lines

Background  

We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines.     

Celecoxib increases retinoid sensitivity in human colon cancer cell lines

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE₂ production was measured with the ELISA assay. The expression of RARβ was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE₂ did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARβ in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE₂ did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARβ and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.     

Effect of selenium on the growth of three human colon cancer cell lines

The effects of selenium were investigated on three human colon cancer cell lines: Caco 2, HRT 18, and HT 29. At low concentrations (10-100 nM), selenium stimulated cell growth in serum-free medium. Thus, selenium is an essential trace element for cell proliferation. At higher concentrations, selenium inhibited cell growth. The rate of 75Se uptake was the same in all of the cell lines studied, but the quantity incorporated differed. GSH-Px activity was dependent on the selenium content of the medium. DNA and protein synthesis paralleled the growth curve. Comparison with the curve of viability revealed that selenium inhibited cell growth in two ways: by inhibiting DNA synthesis, without affecting cell viability, and, at higher doses, by cytotoxicity.     

Isolation and characterization of two novel colon cancer cell lines from Chinese patients

Incidence of colon cancer has increased rapidly in China. Although many colon cancer cell lines have been established previously, most of them were derived from patients from western countries. Epidemiological, clinical, cytogenetic, and molecular biological studies showed that there are considerable differences between Chinese and western countries colon cancer patients. Therefore, establishment of novel colon cancer cell line from Chinese is useful for studying the racial difference of this disease and can be important for studying the pathogenesis of colon cancer in China. In our laboratory, two novel continuous human colon cancer cell lines, SHT-1 and SHH-1, have been established in vitro from Chinese patients, and both cell lines have been passaged for 4 yr, and they have been continuously subcultured with more than 800 population doubling and without signs of senescence. Both cell lines were obtained from primary tumor tissues during colon cancer surgery. Cells grew rapidly with a doubling time of 36–39 h and a plating efficiency of 26–28%. These cells exhibited an epithelial morphology and expressed cytokeratin. Tumor developed in severe combined immunodeficient (SCID) mice 4–6 wk after inoculated subcutaneously with the cultured cancer cells. Karyotypic analysis and comparative genomic hybridization (CGH) analysis in SHT-1 cells revealed a hypertriploid modal number of 76 with numerous numerical and structural abnormalities previously linked to colon cancer. In another cell line (SHH-1), CGH analysis revealed that −1p13 was the only cytogenetic anomaly.     

The comparative influence of anesthetics on the in vitro proton NMR relaxation times in rat liver

To determine the effect of anesthetics on liver relaxation times in rat, two experiments were performed. In the first experiment, normal and protein-depleted rats underwent total hepatectomy under ether anesthesia or following decapitation. In the second experiment, livers were excised from normal rats under ketamine or pentobarbital anesthesia, or following decapitation. Hepatic T1 and T2 were measured for all animals using a RADX 10 MHz spin analyzer. Ketamine produced T1 values significantly different from decapitation. Ketamine, pentobarbital, and ether in normal animals all produced T2 values significantly different from decapitation. It is apparent that anesthetization of rats prior to in vitro measurement of hepatic relaxation times is not equivalent to decapitation; nor are the anesthetics examined equivalent to one another.     

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.     

Localization of cathepsin B in two human lung cancer cell lines

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.     

Characteristics of alpha-adrenoceptors in two human colorectal cancer cell lines.

The DiFi and HT-29 human colorectal cancer cell lines were characterized and compared with respect to binding properties of alpha adrenoceptors present on the cell surface. Both cell lines possessed alpha-1 and alpha-2 adrenoceptors of high affinity; however, DiFi cells were rich in alpha-1 adrenoceptors, whereas HT-29 cells were rich in alpha-2 adrenoceptors. In each cell line, specificity of radioligand binding to alpha-1 or alpha-2 adrenoceptors was proved via competition studies using non-tritiated drugs. We believe this to be the first characterization of alpha-1 adrenoceptors in cell line HT-29 and of alpha-1 and alpha-2 adrenoceptors in DiFi cells. Differences between these cell lines in alpha adrenoceptor expression are discussed in relation to colon carcinogenesis. The high level of alpha-1 adrenoceptors seen in DiFi cells should make this cell line useful in studies of the function and regulation of this adrenoceptor subtype.     

Heterogeneity in tumor cell energetic metabolome at different cell cycle phases of human colon cancer cell lines

In this present study, the efficacy of metabolomics as a tool for tumor cell energetics for in vitro cell cultures was demonstrated with full competence for the first time by elucidating the anabolic and energy-yielding segments of glycolysis and glutaminolysis, which constitute a part of energy metabolism in tumor cells. By synchronizing colon cancer cells SW480 and SW620 in culture, the metabolome specific to cell cycle phases was analyzed using nuclear magnetic resonance spectroscopy. At the G₁/S transition of the cell cycle (i.e. transition from cell growth to duplication of genetic material), the majority of the energy production was realized by glycolysis through a high channeling of glucose carbons towards lactate. During the late S phase, the majority of energy was produced by glutaminolysis through a high channeling of glutamine carbons towards lactate, while the glucose carbons were channeled towards bio-synthetic pathways. These results indicate that the metabolism of proliferating cells is heterogeneous throughout the cell cycle and can be better interpreted on the basis of different cell cycle phases. These findings could be exploited for the development of a tool for tumor diagnosis as well as for targeting tumors.     

Expression of REG protein during cell growth and differentiation of two human colon carcinoma cell lines.

We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.     

Chromosome analysis on two long-term established human colorectal carcinoma cell lines

A chromosomal analysis was carried out on two colorectal carcinoma cell lines (WiDr and COLO 205), which were established 15-20 years ago in the US and were collected by the Cell Bank of the Veterans General Hospital in Taipei. Among the 200 cells counted, 65.5% of WiDr (male) had 68-73 chromosomes, while 74% of the COLO 205 (female) had 70-76 chromosomes per cell. The Y chromosome was absent in the 30 WiDr metaphases analyzed. None of the other chromosomes, including X and the autosomes of both WiDr and COLO 205, revealed such a numerical deficiency. Over half of the autosomes had an average number per cell above 2.0. The existence of 5 or 6 normal homologues for certain autosomes was not rare in either line. Numerous structural abnormal marker chromosomes were present in the cells. As compared with the original chromosome findings which were done over 10 years ago, we noted that the range of chromosome counts was wider and the number of marker chromosomes increased in these long-term cultivated cell lines.     

Sanguinarine causes DNA damage and p53-independent cell death in human colon cancer cell lines

The benzophenanthridine alkaloid sanguinarine has antimicrobial and possibly anticancer properties but it is not clear to what extent these activities involve DNA damage. Thus, we studied its ability to cause DNA single and double strand breaks, as well as increased levels of 8-oxodeoxyguanosine, in human colon cancer cells and found DNA damage consistent with oxidation. Since the tumor suppressor p53 is frequently involved in inducing apoptosis following DNA damage we investigated the effect of sanguinarine in wild type, p53-mutant and p53-null colon cancer cell lines. We found them to be equally sensitive to this plant compound, indicating that cell death is not mediated by p53 in this case. In addition, our observation that apoptosis induced by sanguinarine is initiated very rapidly raised the question whether there is enough time for cellular signaling in response to DNA damage. Moreover, the abundance of double strand breaks is not consistent with only oxidative damage to DNA. We conclude that the majority of DNA double strand breaks in sanguinarine-treated cells are likely the result, rather than the cause, of apoptotic cell death and that apoptosis induced by sanguinarine is independent of p53 and most likely independent of DNA damage.     

Peptidomic analysis of human cell lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.     

Antiproliferative effects of nitrosulindac on human colon adenocarcinoma cell lines

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colon cancer, but their use is limited by toxicity in the gastrointestinal tract. The coupling of a nitric oxide-releasing moiety to NSAIDs strongly reduces these side effects. We demonstrated that the NO-releasing sulindac (nitrosulindac) has much more potent effects on colon adenocarcinoma cell lines compared to sulindac. Moreover, it could inhibit the growth of cells in soft agar experiments, demonstrating the antineoplastic activity at low concentration of nitrosulindac. However, this reduction in the growth of colon cancer cells seemed to be independent of the classical apoptosis pathway and could be explained by a cytostatic effect. Nitrosulindac caused a light perturbation of the cell cycle parameters not linked to a modification of the levels of p21 or the proliferating cell nuclear antigen. Moreover, neither sulindac, nor nitrosulindac, were able to inhibit the NF-kappa B pathway. These data suggested that nitrosulindac could be a better solution compared to other NSAIDs in the treatment of colon cancer.     

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6–18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.     

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.