A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.     

Immunoglobulin production by a human-mouse somatic cell hybrid

The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.     

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

A transformed murine Leydig cell line expresses the ETA receptor subtype

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be <0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (<10 pg/ml). The data from competitive binding experiments with [¹²⁵I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ET_A receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ET_A receptor subtype but do not produce significant quantities of ET-1 under basal conditions.     

The nature of thymidine kinase in the human-mouse hybrid cell

In order to characterize the thymidine kinase in human-mouse somatic cell hybrids derived from mouse parental cells lacking thymidine kinase, we have examined the electrophoretic migration on starch gel and the heat sensitivity of this enzyme in human, mouse, and hybrid cells. The enzyme of hybrid cells migrates similarly to that of human fetal liver and human diploid fibroblasts and faster than that of either L or A₉ mouse cells. It is less sensitive to heat than that of the mouse cells. Therefore, the human group E chromosome provides human thymidine kinase for the hybrid cell. The electrophoresis of thymidine kinase makes possible the search for variants.Aided by U.S.P.H.S. Grant No. HD 00486.     

Establishment of a human leukemia HL-60 cell line that expresses high levels of M-CSF receptors

In Vitro Cellular &; Developmental Biology - Animal -     

A human hepatoma cell line expresses a single-chain form of gamma-glutamyl transpeptidase

Mammalian gamma-glutamyl transpeptidases characterized thus far have been shown to be heterodimeric glycoproteins. The two subunits are derived from a single-chain propeptide which, in the rat kidney, exhibits low transpeptidase activity (less than 2% of the dimeric enzyme). A human hepatoma-derived cell line, Hep G2, expresses relatively high transpeptidase activity. The enzyme is primarily localized on the cell surface and exhibits catalytic properties similar to the dimeric human kidney and lymphoid cell transpeptidase. Significantly, the Hep G2 enzyme, unlike the enzyme from other human tissues, is a single-chain species, Mr = 120,000.     

A human neuroblastoma cell line expresses mu and delta opioid receptor sites

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.     

A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.     

Increased chromosomal radiosensitivity of a Chinese hamster ovary cell line that inducibly expresses the Eco RI restriction endonuclease

We have transfected a Chinese hamster ovary cell line (CHO 6) with a plasmid that inducibly expresses the Eco RI restriction endonuclease gene in the presence of cadmium sulfate (CdSO4). Expression of Eco RI results in DNA double-strand breaks, which can lead to chromosome aberrations. The new line, designated CHO 10, also has a low level of constitutive expression of Eco RI in the absence of CdSO4 without any cytogenetic effect. This suggested that these cells may be efficient at repairing low levels of DNA double-strand breaks. To test this, both cell lines were exposed to ionizing radiation, and aberration yields were analyzed with or without induction of Eco RI. CHO 10 cells showed increased radiosensitivity after G1 irradiation, but after G2 exposure, only doses greater than or equal to 0.4 Gy caused more damage in CHO 10 cells. We conclude that CHO 10 cells can tolerate constitutive expression of Eco RI, but that when the cells are subjected to additional stress, in this case ionizing radiation, they become very sensitive to DNA double-strand breaks.     

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

An immunocytochemical screening of human-mouse cell hybrid colonies expressing a specific human gene

An immunocytochemical method has been devised which allows the screening of a large number of human × mouse cell hybrid colonies for the retention of a specific human chromosomal gene and the presence of its translation product. The glycolytic enzyme phosphoglucose isomerase (PGI; d-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was chosen as a marker which is known to be controlled by the gene on human chromosome 19. The technique involves three steps: (i) immobilization of growing cell colonies in agar gel containing antibody that specifically reacts with human-type PGI; (ii) lysis of the embedded cells with Triton X-100 to release enzyme antigens and precipitate as an immune complex; and (iii) visualization of the antibody-fixed enzymes by histochemical activity staining. Human PGI activity released from a colony consisting of as few as eight cells generated an adequate signal. Variation of intensity was noticed and attributed to gene dosage in individual cells. The percentage of human PGI-positive colonies in each of nine independent hybrid lines estimated by this method generally paralleled the frequency of retention of human chromosome 19 determined by conventional karyotyping. The technique can be applied to many other markers and be used as a half-selection system in combination with the replica plating method.This work was supported by National Institutes of Health Grant GM 24375. N.S. is the recipient of American Cancer Society Junior Faculty Research Award JFRA-9.     

The cloned cell line L10A2.J expresses natural cytotoxic activity

The analysis of natural cytotoxicity (NC) has been hampered by the lack of cloned NC effectors. In studies reported here we show that the cloned cell line L10A2.J expresses properties similar to those of splenic NC effectors. L10A2.J cells lyse NC-sensitive targets, but do not lyse NC-resistant targets which are sensitive to lysis by natural killer (NK) or cytotoxic T lymphocytes. The mechanism by which L10A2.J cells lyse NC-sensitive targets is similar to the lytic mechanism of splenic NC effectors in that both result in the release of 51Cr from targets with a lag of 5-7 hr after effectors and targets are mixed. In addition, inhibition of protein synthesis during the in vitro assays of NC or L10A2.J lytic activity causes some NC-resistant targets to become sensitive to lysis by both NC and L10A2.J effectors. The only functional difference detected between L10A2.J and splenic NC effectors is in their recognition of targets. While L10A2.J and splenic NC effectors recognize many of the same targets (NC resistant and NC sensitive), L10A2.J, unlike splenic NC effectors, does not recognize the NK-sensitive cell line YAC-1.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.     

稳定表达β-synuclein的SH-SY5Y细胞株的构建

目的构建稳定表达β-synuclein的SH-SY5Y细胞株。方法利用脂质体转染技术将质粒pcD-NA3.1-β-synuclein转染SH-SY5Y细胞,通过Zeocin进行抗性筛选。采用原位免疫荧光、免疫组织化学染色及Western blot杂交检测β-synuclein在SH-SY5Y细胞中的表达情况;通过MTT比色法检测β-synuclein稳定表达对SH-SY5Y细胞增殖的影响。结果原位免疫荧光、免疫组织化学染色及Western blot检测结果显示β-synuclein在SH-SY5Y细胞中的表达水平较对照组明显升高;稳定表达β-synuclein的细胞增殖程度较对照组明显增高(P<0.05)。结论β-synuclein在SH-SY5Y细胞中稳定表达,为后续的研究提供有用的细胞模型。为进一步研究β-sy-nuclein对帕金森病发病神经保护作用的研究奠定了良好的基础。