Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The COMMA-D cell line derived from mammary epithelial cells of midpregnant mice was shown previously to be heterogeneous as determined by phase-contrast microscopy, immunocytochemical staining, DNA content, and oncogenic potential (K.D. Danielson et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3756; D. Medina et al. (1986) J. Natl. Cancer Inst. 76, 1143). Clonal subpopulations of COMMA-D cells have now been isolated by both transfection and selection using a dominant-selectable gene transfer vector and by limiting dilution. Despite their clonal origin, these subpopulations in many cases retained the heterogeneity of the parental COMMA-D line. Of 18 clonal lines assayed, only 5 were able to express beta-casein mRNA. Pooled populations of G418-resistant cells expressed substantially higher levels of beta-casein mRNA than the clonal lines. One of the expressing clonal lines, BNW-7, was characterized further, using immunocytochemical techniques. Approximately 10% of BNW-7 cells expressed casein under the appropriate hormonal and cell-substratum conditions by indirect immunofluorescent staining. Casein immunoperoxidase staining of BNW-7 cells on floating collagen gels revealed that casein-producing cells were localized in small alveolar structures, which were formed in a non-hormone-dependent fashion. The cells in these alveolar structures were cuboidal with basally located nuclei, expressed keratin intermediate filament proteins preferentially, and comprised approximately 18% of the total cells. Cells elsewhere on the surface of the gel displayed a flattened morphology, and expressed vimentin intermediate filament proteins preferentially. A proportion of COMMA-D cells, therefore, appeared to have some of the characteristics of mammary stem cells, and retained the ability to differentiate and form phenotypically heterogeneous cell populations in vitro.
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PMID:A clonal derivative of mammary epithelial cell line COMMA-D retains stem cell characteristics of unique morphological and functional heterogeneity. 245 48

Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated c-rasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fos/FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express gamma-glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since gamma-glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated c-rasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.
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PMID:Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene. 247 37

The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.
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PMID:HPV-18 immortalization of human keratinocytes. 247 70

Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.
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PMID:Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes. 768

Human papillomavirus (HPV) has been implicated in the etiology of oral and cervical cancers. Normal oral epithelial cells at passage two were infected with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. The G418-selected cells that were immortalized and express HPV 16 E6/E7 have been in culture for over 4 years and 350 passages. In contrast, the normal oral epithelial cells did not survive the culture environment beyond 7 to 9 passages. Fifteen clones were selected from the pooled population. By Northern blot analysis all clones demonstrated the presence of E6solidusE7 genes. Keratin expression of both normal and immortalized oral epithelial cells was studied in organotypic culture. Both cell types were positive with antibodies AE1, AE3, and 34BE12. Both were focally positive with AE8, which stains for keratin 13 (specific for oral and esophageal epithelial cells). The normal control cells were focally positive for filaggrin, while the HPV 16-immortalized cells (IHGK cells-immortalized human gingival keratinocytes) were negative. The IHGK cells were strongly positive with KS19.1, which stains the embryonal keratin K19, an indicator of premalignant or malignant changes, while the normal control cells were only lightly and focally positive. In conclusion, we present an oral epithelial cell line successfully immortalized with HPV E6/E7 which will facilitate further research on the involvement of HPV in oral carcinogenesis.
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PMID:HPV immortalization of human oral epithelial cells: a model for carcinogenesis. 866 Sep 52

We have got the humanized antibody with high affinity and specificity against keratin by phage antibody library technology. To improve protein yields and get high affinity and specific anti-keratin antibody, we chose to express it in Pichia pastoris. Anti-keratin ScFv gene from plasmid p3MH/ScFv was subcloned into vector pPIC9K. After confirmed by DNA sequence analysis, the recombinant plasmid pPIC9K/ScFv was transduced into the genome of GS115 P. pastoris. Mut(s) multiple insert transformants were screened by G418 and induced by 5 mL/L methanol to express soluble ScFv. After 6 days of methanol induction, anti-keratin ScFv was efficiently secreted into the medium. Western blot and ELISA assay proved the expressed protein had specific keratin-binding activity. After purification, we examined its effect on cultured keratinocytes by Cell cycle analysis, Which indicated that human ScFv against keratin 17 can inhibit the proliferation of keratinocytes by influence the synthesize of keratinocyte DNA. The successful expression of anti-keratin ScFv in P. pastoris laid a solid foundation for its further application.
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PMID:Secretory expression of human ScFv against keratin in Pichia pastoris and its effects on cultured keratinocytes. 1893 42

The insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the 6 members of the IGFBP family and is involved in the regulation of cell growth, apoptosis, and other IGF-stimulated signaling pathways. To determine the significance of IGFBP-5 in the Inner Mongolia Cashmere goat (Capra hircus), a hair follicle-specific expression vector of IGFBP-5, pCDsRed2-K-IGFBP5 (6.7 kb), was constructed by cloning IGFBP-5 downstream of the keratin-association protein (KAP)6-1 promoter and inserting this fragment into pCDsRed2, which contains a red fluorescent protein (DsRed) expression unit. Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by using Lipofectamine(TM) 2000. Cell clones that stably expressed red fluorescence were obtained after selection with Geneticin (G418). The transgene in the cell clones was examined by polymerase chain reaction to verify that exogenous DNA (pKAP6-1 and IGFBP-5) had integrated stably into GFb cells. These data suggest that this method can be used for the construction of a hair follicle-specific expression vector for functional genetic analyses and for obtaining stable transfection donor cells for nuclear transfer.
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PMID:Stable transfection and identification of a hair follicle-specific expression vector of IGFBP-5 in goat fetal fibroblasts. 2466 76