Magnetic selection of transiently transfected cells

An efficient and rapid method for selecting transiently transfected cells is described. A plasmid encoding for a neural cell-specific surface marker is co-transfected into mammalian cells along with the gene of interest. After uptake and expression of these two plasmids, the transfected cells are immuno-adsorbed to magnetic beads pre-coated with antibodies against the surface marker. These immuno-complexes are then isolated by means of a strong magnet. In a single round of magnetic selection, we were able to enrich the cell population more than 7-fold for a co-transfected reporter. These specifically selected cells can now be used for either further cultivation or for immediate analysis. This method has been shown to be effective on HeLa and on CV-1 cells and is expected to give similar results on any other transfectable, non-neuronal cell lines.     

Detection and quantification of apoptosis in transiently transfected adherent cells

Apoptosis is a highly conserved form of cell death present in all eukaryotic cell types and controlled by multiple genes. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. It is particularly difficult to reliably assay apoptosis of adherent cells. We describe a new, rapid and reliable flow cytometric method which can be used for quantifiying apoptosis in a sub-population of transiently transfected adherent cells. This technique is based on the detection of transfected cells and the apoptotic sub-population by immunofluorescence and Annexin-V labelling, respectively.     

Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.     

Inhibition of hepatocellular carcinoma development in hepatitis B virus transfected mice by low dietary casein

In a comprehensive human ecological study, primary liver cancer has been shown to be highly significantly associated with 1) the prevalence of persistent infection with hepatitis B virus (HBV) and 2) plasma cholesterol concentrations that are, in turn, associated with the consumption of animal based foods. In rat studies, aflatoxin-induced hepatocellular carcinoma is substantially prevented by decreasing the intake of animal based protein (casein), a hypercholesterolemic nutrient. Thus the development of primary liver cancer associated with persistent HBV infection or with aflatoxin exposure may be controlled by reduced intake of animal-based proteins. Transgenic mice transfected with an HBV gene fragment containing the viral transactivator of hepatis B virus, HBx, which induces the formation of hepatocellular carcinoma, were used to examine the ability of dietary casein to modify tumor formation. Reducing the concentration of dietary casein to 6% from the traditional level of 22% markedly inhibited (by 75%) hepatic tumor formation in these transgenic mice. Tumor development also was substantially altered by interchanging dietary casein concentration well after tumor development had begun (at 8 months), increasing by 173% from the expected yield when casein intake was increased and decreasing by 99% when casein was reduced. These findings suggest that the development of liver tumor formation among individuals persistently infected with HBV may be controlled by minimizing or eliminating the intake of animal protein-based foods.     

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.     

Polarized secretion of apoA-I and apoA-II by transfected MDCK cells

Apolipoproteins (apo) are secreted preferentially from the basolateral surface of hepatocytes and enterocytes. The polarized secretion of proteins is either mediated by a protein-dependent sorting signal or by a cell-dependent default pathway. In order to determine the mechanism for the polarized secretion of apolipoproteins, we examined the secretion of apoA-I and apoA-II in transfected Madin-Darby canine kidney (MDCK) cells. Transfected MDCK cells and Caco-2 cells were grown as a polarized monolayer on tissue culture inserts, which separate an upper apical compartment from the lower basolateral compartment, and the secretion of apoA-I and apoA-II into the apical and basolateral compartments was quantitated by immunoprecipitation. Caco-2 cells almost exclusively secreted apoA-I and apoA-II basolaterally, with an apical to basolateral ratio of 18:82 for apoA-I, and 11:89 for apoA-II. In contrast, transfected MDCK cells secreted significant amounts of apoA-I and apoA-II into both compartments, but with a bias toward apical secretion and an apical to basolateral ratio of 66:34 and 68:32, respectively. The polarized secretion of MDCK cells was not due to transcytosis, diffusion, or differential recovery. As assessed by density gradient ultracentrifugation, apoA-I and apoA-II secreted from either the apical or basolateral surface were relatively lipid-poor. Overall, these results suggest that the polarized secretion of apoA-I and apoA-II does not occur by a protein-dependent sorting signal, but by a cell-dependent default pathway that leads to preferential basolateral secretion by Caco-2 cells and both apical and basolateral secretion in MDCK cells, but with a bias toward apical secretion.     

Assembly of hair keratins in transfected epithelial cells

To define the interactions required for the filament assembly of differentiation-specific keratins, active copies of mouse hair keratin mHa1 and mHb4 genes were introduced into a rat kangaroo kidney epithelial cell line (PtK2) and a rat stratified squamous epithelial cell line (rat epidermal keratinocyte). In PtK2 transient transfectants, when introduced individually or in combination, mHa1 and mHb4 formed aggregates of ring-like structures of various sizes at the perinuclear region with no evidence of organization into a keratin network. These aggregates altered the distribution of the endogenous keratins and vimentin. In most of the cells carrying the ring-like structures of mHa1 and mHb4 around the nucleus, the endogenous keratin network collapsed and localized around the nucleus. Furthermore, the densely accumulated endogenous keratin surrounded the ring-like aggregates with partial co-localization. However, when transfected into the rat epidermal keratinocytes, mHa1 and mHb4 were able to co-localize with the well-developed cytoskeleton of endogenous keratins. These results showed that, in contrast to keratin pairs K5/K14 and K8/K18, the mHa1/mHb4 pair is unable to develop an extensive keratin network on its own and that there are possible differential abilities among these hair keratins and other keratins to form well-developed cytoplasmic networks.