High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

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.     

Adaptive response induction and variation in human lymphoblastoid cell lines

Adaptive response is a term used to describe the ability of a low, priming dose of ionizing radiation to modify the effects of a subsequent higher, challenge dose, but it has been observed to be highly variable in both presence and magnitude. To examine this variability, 10 human lymphoblastoid cell lines were screened for adaptability to 137Cs radiation by determining the frequency of micronuclei in binucleated cells. Of these, six adapted, three did not adapt and one was synergistic. The assay was then repeated on each of the cell lines to test for reproducibility. Five cell lines showed the same result both times; four of these adapted and one did not.To determine whether fluctuations in the cell cycle distribution in the irradiated population of cells could alter the adaptive response, and therefore explain some of the observed variability, two of the cell lines were tested for adaptation after enriching the population, by synchronization, for a given cell cycle stage. In both cell lines, the direction of the response was altered when the distribution of cells within the cell cycle was changed, suggesting that the adaptive response can be affected by cell cycle stage at the time of irradiation.     

Clinical drug response can be predicted using baseline gene expression levels and in vitro drug sensitivity in cell lines

We demonstrate a method for the prediction of chemotherapeutic response in patients using only before-treatment baseline tumor gene expression data. First, we fitted models for whole-genome gene expression against drug sensitivity in a large panel of cell lines, using a method that allows every gene to influence the prediction. Following data homogenization and filtering, these models were applied to baseline expression levels from primary tumor biopsies, yielding an in vivo drug sensitivity prediction. We validated this approach in three independent clinical trial datasets, and obtained predictions equally good, or better than, gene signatures derived directly from clinical data.     

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.     

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.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Polymorphism of the thymidylate synthase gene and thymidylate synthase levels in colon cancer cell lines and different tissues of colorectal cancer patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Structure and behavior of rat primary and secondary Schwann cells in vitro

The structure and motility of isolated rat primary (I) Schwann cells (SC) have been compared to that of subcultured (II) SC during and after mitotic stimulation. I SC contain myelin components which persist for 2 weeks in serum-free medium while they rapidly disappear in medium containing serum and high glucose concentration. These components were never detected in II SC. Both I SC and II SC after their mitotic phase are spindle-shaped, contain many intermediate and actin filaments, have no basement membrane but show intense migratory and undulatory activities. Rare fibroblasts in I cultures are recognized by their extremely variable shape, the presence of Thy 1.1 antigen in their membrane and their intense edge ruffling alternating with abrupt translocation. In contrast, I SC movements consist of intracellular translocation of nuclei along SC processes, which retract and extend constantly, and in slow rhythmic undulation episodes (2.3 ± 0.2/min) alternating with migration at 135 ± 50 μ/h. The total number of these episodes per day in serum-free medium is rigorously identical for different cells (166.3 ± 0.2) and this uniformity of frequency suggests a genotypic basis. Cycles, consisting of an undulation episode followed by a resting interval, have mean durations of 8.6 ± 4.1 min and a sharp peak of occurrence at 6 min, with exponential distribution of the longer periods. Motility of II SC is considerably inhibited during mitotic stimulation by cholera toxin and a pituitary extract while SC phenotype has changed to a flat multipolar cell with prominent Golgi and ribosomes. Migration is reduced to 24 ± 2 μ/h and only 2% of the SC show pulsations of the same periodicity as the I SC undulations. A dramatic increase in pulsation frequency occurs 6–12 h after removal of mitogenic factors when 80% of II SC start pulsating twice as fast for 2–3 days. When mitoses cease, SC quickly recover their SC phenotype with rhythmic undulations while migration speed increased to 92 ± 20 μ/h. Thus, in spite of dramatic modification of shape, structure and behavior during mitotic stimulation, SC subsequently recover their unique motility pattern which might be essential for their myelinating function.     

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

Expression and distribution of HSP27 in response to G418 in different human breast cancer cell lines

Heat shock proteins (HSPs) play an important role in folding, intracellular localization and degradation of cellular proteins. However, the cellular role of HSP27 is not completely understood. The conflicting results have been reported regarding stress-induced nuclear translocation of HSP27. In this study, human breast cancer cells transiently and stably expressing HSP27–EGFP chimera were utilized to observe the intracellular localization of HSP27. The data show that the transient and stable expression of HSP27–EGFP displayed distinguishingly cellular localization. The nuclear translocalization of HSP27–EGFP was correlated with the presence of G418. Experiments carried out with different human breast cancer cell lines revealed clearly different distribution patterns of endogenous HSP27. The subcellular distribution of endogenous HSP27 appeared diffuse throughout the cytoplasm in MDA435 cells. In MCF-7 and SKBR3 cells, the accumulation of the protein was distinctly seen along the cell membrane and around nucleus. Moreover, the nuclear translocation of endogenous HSP27 was stimulated by G418 only in MDA435 cells, but not in MCF-7 and SKBR3 cells. Overexpression of HSP27 has been associated with resistance to cisplatin and doxorubicin. The correlation of the expression pattern of HSP27 with the drug resistance may need to be investigated. Further studies on the intracellular function of HSP27 may take into account its interaction proteins in the cells. It may provide useful information for the identification of sensitivity of carcinoma cells to the chemotherapeutic drugs and development of more specific agents to circumvent HSP27.     

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.     

Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

Deoxyribose protects against rapamycin-induced cytotoxicity in colorectal cancer cells in vitro

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.     

Nuclear expression of thymidylate synthase in colorectal cancer cell lines and clinical samples.

Thymidylate synthase (TS) [TYMS; OMIM reference number (188,350)] is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation, and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS also was seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-fluorodeoxyuridine were fractionated and examined by Western blotting. Interestingly, both free TS and the ternary complex of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS, and HT-29, RKO, and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.