Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.     

Expression of mouse::human immunoglobulin heavy-chain cDNA in lymphoid cells

A chimeric mouse variable::human constant immunoglobulin heavy-chain gene was expressed in transfected mouse Sp2/0 cells. The chimeric immunoglobulin genes were integrated in tandem in the genome of stably transformed cells. These integrated gene copies were amplified by selection with a second drug marker. The gene amplification led to an increase in the expression of chimeric heavy-chain protein. The level of gene expression appears to be related to the site of integration; a few gene copies in one transfectant can yield as much heavy-chain protein as many copies in a second transfectant. In addition, we found that an adventitious oligo(C) sequence, introduced by our method of gene construction at a site located 8 nt residues downstream from a splice acceptor, can apparently direct splicing towards a cryptic splice acceptor downstream from the oligo(C).     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells.

Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF-10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF-10A) and oncogene transformed (MCF-10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF-10M, MCF-10A, and MCF-10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF-10M, but not in MCF-10A and MCF-10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.     

Regulated expression of human atrial natriuretic polypeptide gene in mouse L cells

To investigate whether the human atrial natriuretic polypeptide (hANP) gene is responsive to glucocorticoid, we co-introduced the hANP gene (with SV40 enhancer) with HSV-tk gene into mouse tk- L cells. The transformants with hANP gene with SV40 enhancer expressed hANP specific RNAs. The administration of 1 microM dexamethasone reduced the expressed hANP specific RNAs, especially those that had a physiological initiation site. These results suggest that the hANP gene is really a glucocorticoid responsive gene and may be negatively regulated by glucocorticoid.     

Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells

Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.     

Expression of cotransferred genes in mouse L cells

The Herpes simplex virus (HSV) thymidine kinase (TK) gene was introduced into mouse L cells by cotransformation with the G418 resistance gene on plasmid pSV2neo.d5 and selection for antibiotic resistance. Fifty nanograms each of the TK gene and pSV2neo.d5 were mixed with 10 μg of carrier DNA and precipitated onto cells. Of 18 antibiotic-resistant colonies analyzed, 15 contained 1–5 copies of the TK gene and 13 of the 15 were phenotypically TK positive. These data demonstrate that it is possible to introduce small numbers of copies of cotransferred genes into cultured cells and that in most cases the genes are integrated in a manner compatible with expression.     

Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA

Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype.     

Expression of transfected human CuZn superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.     

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.     

Expression in mouse L cells of mos-related ORAgp5 locus cloned from the human genome]

The recombinant locus ORAgp5 containing the regions homologous to protooncogene mos was previously cloned from the human genome. In order to monitor the expression of this human genome region, we have transfected the recombinant phage gp5 into mouse LMtk cells. One of these transfectants contained several copies of gp5. ORAgp5 sequences were found to be expressed in 1.5 kb RNA from this clone.     

Expression of human cardiac actin in mouse L cells: a sarcomeric actin associates with a nonmuscle cytoskeleton

A cloned human cardiac actin gene, introduced into mouse Ltk- cells, is expressed in several thymidine kinase (tk)-positive cotransfectants. The clones not only produce authentic polyadenylated human cardiac actin mRNA but also synthesize human cardiac actin protein. The cardiac actin protein, normally found only in myofibrils, is stably accumulated at a high level, about one-third that of the endogenous mouse beta-actin. Furthermore, this sarcomeric protein partitions between the Triton X-100 insoluble and soluble phases to the same extent as the endogenous beta-actin. This suggests that a sarcomeric actin can participate in the formation of Triton X-100-insoluble cytoskeletal structures.