Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.     

Fixation of the unmethylated or the 5''-CCGG-3'' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns.

The late E2A promoter of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promoter. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W. Doerfler, Proc. Natl. Acad. Sci. USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci. USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promoter-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promoter methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promoter controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promoter controls the gene for neomycin phosphotransferase. The pAd2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuration. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAd2E2AL-CAT construct, the late E2A promoter remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promoter remained almost completely methylated. In five cell lines, the E2A promoter sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylations were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promoter, after this promoter was fixed by integration in the mammalian genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.     

Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.     

An optimum dose of c-H-ras is a prerequisite for hormone-dependent conversion of a cell between cancerous and normal states in tissue culture.

We have established a few cell lines which can be reversibly converted from cancerous to normal and vice versa by the addition to, or removal from the culture medium of glucocorticoid hormone. These cell lines were derived from mouse NIH 3T3 cells and possessed the integrated gene pairs on chromosomes, which are composed of human mutated c-H-ras fused with mouse mammary tumour virus long terminal repeat and E. coli xanthine-guanine phosphoribosyltransferase gene with the SV40 promoter. We have characterised these cell lines in order to elucidate an essential requirement for the conversion of the state of a cell. It was found that the presence of at least two to three copies of the gene pair per diploid genome are essential. An approximate threshold level of c-H-ras 1.6 kb RNA required for reversible conversion was estimated.     

StARD13(Dlc-2) RhoGap mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.