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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.
Mol Cell Biol 1989 Apr
PMID:A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. 265 95

An hepatocyte transplantation-gene transfer protocol has been developed whereby liver cells containing an expressing NeoR gene can be successfully implanted in vivo. Adult primary cultures of rat hepatocytes, after infection with the retroviral vector N2, were grown on a floating solid support (coated with purified collagen IV) in a serum-free hormonally defined medium designed for hepatocytes that also contained G418. Under these conditions, normal adult hepatocytes expressing the NeoR gene could be grown to high density. The solid supports holding the gene-engineered hepatocytes were then implanted into adult rats into subcutaneous and intraperitoneal sites. After one to two weeks, the supports were removed and shown to still contain the gene-engineered hepatocytes expressing the NeoR gene. These results suggest that cells from solid organs, such as the liver, are potential targets for gene transfer and expression studies in vivo.
Somat Cell Mol Genet 1989 May
PMID:Gene expression in implanted rat hepatocytes following retroviral-mediated gene transfer. 265 27

To analyze the mechanism of neoplastic transformation of rodent diploid cells by ras and myc oncogenes, human EJ c-Ha-ras and mouse c-myc second and third exons promoted by SV40 promoter were connected to pSV2neo and pSV2gpt, respectively. Mouse and rat primary fetal cells cotransfected with both genes formed transformed and nontransformed colonies in a medium containing G418 and mycophenolic acid (MPA). The proportion of transformed colonies in the total G418/MPA-resistant colonies decreased dependent on the stage of the gestation period of rat fetuses from which primary cells had been obtained. Analysis of randomly isolated colonies showed that the transformed colonies had a high copy number and high amount of expression of the introduced genes, were anchorage independent, and were tumorigenic in nude mice. On the other hand, the nontransformed colonies had a low copy number, low amount of expression, and no tumorigenicity. This contrast indicated not only that the activated Ha-ras and myc oncogenes had been integrated, but also that the amplification or overexpression (or both) of these genes was required for the rodent diploid cells to be transformed. We conclude that early-stage rat fetal cells might have endogenous factors that promote cell transformation. Alternatively, late-stage cells might have factors that suppress cell transformation by activated Ha-ras and myc oncogenes.
Mol Carcinog 1989
PMID:Cotransfection of plasmids with ras and myc oncogenes to diploid cells derived from rodent fetuses: alteration of neoplastic transformation frequency depending on the gestation period. 267

In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 microns sequence yield stable transformants. We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.
Mol Microbiol 1989 Aug
PMID:Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii. 269 36

It has been suggested that transforming growth factor-alpha (TGF-alpha) is a mitogenic autocrine growth factor for human breast cancer cells, responsible for mediating the mitogenic effects of 17 beta-estradiol (E2) in responsive cells. To test this hypothesis we have introduced eukaryotic expression vectors directing the expression of TGF-alpha mRNA into E2-responsive MCF-7 human breast cancer cells. Transfected cells produce levels of TGF-alpha equivalent to or greater than those produced by both E2-stimulated MCF-7 cells and hormone-independent MDA-MB-231 cells. One transfected clone (H8) secretes sufficient TGF-alpha to fully down-regulate EGF-R expression. However, both of the transfected clones that constitutively secrete elevated levels of TGF-alpha (A8 and H8) respond to E2 stimulation in vitro by increasing the rate of cellular proliferation and inducing PGR synthesis. The basal proliferative capacity of H8 and A8 cells is equivalent to that of the parental cells and to cells transfected only with the G418 (neomycin) resistance gene. Furthermore, the TGF-alpha cDNA-transfected clones do not form tumors in ovariectomized athymic nude mice without E2 supplementation. Thus, the precise role of TGF-alpha in mediating either the in vivo or the in vitro mitogenic effects of E2 in MCF-7 human breast cancer cells remains unclear. While TGF-alpha expression may be essential, it is not sufficient alone to induce the fully E2-independent phenotype. Thus, TGF-alpha may function in combination with other E2-induced growth factors to control breast cancer proliferation and tumorigenesis.
Mol Endocrinol 1989 Feb
PMID:The effects of a constitutive expression of transforming growth factor-alpha on the growth of MCF-7 human breast cancer cells in vitro and in vivo. 271 Jan 38

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfection, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418-resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo-tagged human chromosome 11 in all of the five SiHa-microcell hybrids. Two SiHa-microcell hybrids that contained a single copy of neo-tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa-11) were suppressed; four of the five SiHa-11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa-11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor-suppressor gene (or genes) for human cervical tumors.
Mol Carcinog 1989
PMID:Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa. 273 Jul 61

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 Jul
PMID:Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins. 274 27

To determine whether the enhanced expression of transforming growth factor alpha (TGF alpha) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGF alpha cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGF alpha mRNA expression by northern blot analysis, TGF alpha secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGF alpha-transfected NOG-8 clones expressed high levels of a specific TGF alpha mRNA, secreted elevated levels of TGF alpha into the culture medium (177-595 ng/10(8) cells/48 h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGF alpha peptide was able to inhibit colony formation in soft agar by TGF alpha-transfected NOG-8 clones that produced high levels by TGF alpha. This inhibition suggested that TGF alpha acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGF alpha, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGF alpha cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.
Mol Carcinog 1989
PMID:Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA. 278 19

We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.
Somat Cell Mol Genet 1987 Sep
PMID:Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers. 282 33

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.
Mol Cell Biol 1987 Aug
PMID:Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes. 282 7


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