A colonic tissue architecture assay applied to human colon carcinoma cells

Summary A two-component tissue architecture assay system has been devised that tests the ability of human colon carcinoma cells to conform to the specific three-dimensional cell-cell and cell-substratum interactions characteristic of normal colonic tissues. Dissociated fetal rat colonic cells (FRCC) were allowed to reaggregate in suspension with or without the addition of different proportions (0.1%, 1%, and 10% of the total cells) of the human colon carcinoma cell lines, SW-1222 and LS-174T. Cellular aggregates obtained after 36 hours’ incubation exhibited cell sorting by the formation of recognizable epithelial colonic crypt-like structures with glandular lumens in a mesenchyme-like background. Carcinoembryonic antigen (CEA)-positive SW-1222 cells in 10% mixed aggregates were organized into numerous well-formed glandular structures with a polarized apical distribution of CEA. LS-174T cells, on the other hand, were self-sorted but structurally disorganized with a continuous cell surface CEA distribution. Pure FRCC and mixed aggregates were implanted under the kidney capsules of Swiss nu/nu (nude) or CD-1 nu/nu mice and allowed to grow for a period of 7–10 days. Whereas the normal FRCC readily formed colonic tissue, the SW-1222 cells exhibited a capacity for differentiation into colonic crypts which became progressively less normal and more tumor-like as the proportion of carcinoma cells in the aggregates was increased. The LS-174T cells demonstrated poor differentiation at all concentrations. Cell surface levels of CEA and the CEA family member nonspecific crossreacting antigen (NCA), both overexpressed in colon cancer, were higher in LS-174T than in SW-1222 cells, whereas family member biliary glycoprotein (BGP), downregulated in colon carcinoma was higher in the SW-1222 cells. These results thus support the suggestion that deregulated expression of CEA family members can be involved in the ability of colonocytes to differentiate and conform to normal tissue architecture as assessed by the assay. The assay is therefore amenable to genetic analysis of normal and perturbed architectural phenotypes. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. C. I. is a recipient of a studentship from “le Fonds pour la Formation de Chercheurs et l’Aide à la Recherche.”     

Polyplex‐mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age‐related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome‐mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex‐mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20\% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex‐mediated gene transfer, but stable integration does occur at low frequencies with and without selection. J. Cell. Biochem. 76:153–160, 1999. © 1999 Wiley‐Liss, Inc.     

Surfactant-mediated gene transfer for animal cells

A commercially available cationic surfactant, dimethyl-dioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. DDAB easily dissolved in water at 60 °C and formed lipid vesicles at room temperature. The lipid vesicles showed very low cytotoxicity compared with other cationic surfactants. After the lipid vesicles were mixed with plasmid DNA solution, the solution was added to mammalian cells. The addition of a nonionic surfactant (Tween 80) to the cationic lipid vesicles at the weight ratio of 1:1 enhanced transfection efficiency. Adding more or less than the optimal amounts of DNA and lipid vesicles resulted in decreased transfection efficiency. With the optimal amounts of DNA (pCMVβ) and lipid vesicles, about 90–95% of CHO-K1 and BHK-21C13 cells transiently expressed β-galactosidase activity 24 h after transfection. By this procedure, stable transformants around 10⁵ cells corresponding to 10% efficiency could be obtained by one batch transfection. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.     

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

The ability to propagate under anaerobic conditions is an essential and unique trait of brewers or bakers yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen.Communicated by C. P. Hollenberg     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology,agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer(MGCMGT) or female germ cell mediated gene transfer(FGCMGT) technique.Sperm-mediated gene transfer(SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been exten...     

Adoptive transfer of natural killer cells in combination with chemotherapy improves outcomes of patients with locally advanced colon carcinoma

Background

Despite the availability of multiple treatment strategies, patients with advanced colon carcinoma (CC) have poor prognoses. The aim of this study was to evaluate the efficacy and safety of natural killer (NK) cell therapy in combination with chemotherapy in patients with locally advanced CC.

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH₂)₁₀] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6^? and Lipofectin^?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

Mechanism of taurine-induced apoptosis in human colon cancer cells

Taurine （Tau） has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA （p53-upregulated modulator of apoptosis） plays an important role in the process of apoptosis induction in a variety of human tumor ceils in both p53- dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 （mutant p53） and LoVo （wild-type p53） with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell fines. Meanwhile, we also observed the increased expression of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tauinduced apoptosis pathway in human colorectal cancer ceils. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.     

Phenotypic changes and gene expression in human colon mucosal epithelial cells upon transfection of a SV40 DNA-GPT recombinant

Summary Phenotypic changes (increased longevity, decreased growth factor requirements, altered cell surface features, growth in semisolid agarose, and SV40 T antigen expression) suggesting in vitro transformation were displayed by human normal colon mucosal epithelial cells transfected with pSV3gpt, a pBR322 recombinant containing the SV40 “early” T antigen coding region and the dominant selectable marker bacterial gene, xanthine-guanine phosphoribosyltransferase. In contrast, control cultures which received neither DNA nor the recombinatn pSV2gpt (which is identical to pSV3gpt but lacks the SV40 T antigen region) were not phenotypically altered.     

Electrophoretic analysis of four high molecular weight sialoglycoproteins produced by metastatic human colon carcinoma cells

We have found that polyacrylamide gel electrophoresis in 3% gels in the presence of sodium dodecyl sulfate is suitable for the separation of cellular glycoproteins having molecular weights ranging from 200,000 to 1,000,000. The gels secured on a rigid support (Gelbond) allow blotting techniques with lectins and antibodies for the detection of glycoproteins. Using these methods we have separated lysates of HT-29 human colon carcinoma cells and detected at least four distinct high molecular weight sialoglycoproteins having molecular weights of 900,000, 740,000, 560,000, and 450,000. The expression of the 900,000 component, as revealed by wheat germ agglutinin binding, was much higher in a subline of HT-29 cells established from liver metastases in a nude mouse than it was in the parental cells. The relative intensity of wheat germ agglutinin binding to these four sialoglycoprotein components differs depending upon their growth phase in vitro. These glycoproteins were also detectable by the binding of peanut agglutinin, provided the glycoproteins were previously treated in the gels with mild acid to remove the sialic acid from their carbohydrate chains, suggesting that mucin-type carbohydrate chains are present on these glycoproteins. The same set of glycoproteins can be detected by metabolic labeling of the cells with [3H]glucosamine in tissue culture. Very similar glycoprotein profiles are revealed by metabolic labeling of fresh colon carcinoma tissues with [3H]glucosamine in vitro.     

Normal human colon cells suppress malignancy when fused with colon cancer cells

Summary Normal human colon mucosa cells and cells obtained from histologically normal tissues near that cancer were fused with human colon cancer cells. Resultant hybrid populations of normal and malignant cell fusions behaved as nonmalignant cells in culture, were unable to grow in soft agar, did not express tumor-associated antigens, and were nontumorigenic in nude mice. Autofusion of the cancer cell population led to a phenotype intermediate between normal and malignant cells. That is, the cultures had a much lower plating efficiency in soft agar, and the tumors had a longer latency and slower growth rate in nude mice. This is the first cell culture system to demonstrate that normal epithelial cells can suppress malignancy of their autologous cancer cells, and is a prelude to more extensive studies of genetic events involved in malignant conversion of human colonic epithelium. This study was supported by The University of Texas Health Science Center at San Antonio Center for Human Cell Biotechnology and a graduate student stipend (T. J.) from the Department of Cellular and Structural Biology.     

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.     

Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: Absence of autocrine effects

Summary We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of [³H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [³H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed. Supported by grants from the National Institutes of Health, Bethesda, MD (PO1 DK 35608 and CA 38651, and a grant from the American Cancer Society, Washington, DC (PDT-220).