Modulation of HeLa cell growth by transfected 7SL RNA and Alu gene sequences.

Alu and 7SL RNA gene sequences were tested for the potential to regulate mammalian cell growth by introducing these sequences into HeLa cells in a coupled DEAE-dextran transfection/affinity cell sorting system. Both Alu and 7SL RNA genes mediated inhibition of [3H]thymidine and [35S]methionine incorporation in recipient cells. In addition, short term growth curves were performed on calcium phosphate/DNA cotransfected, affinity-purified cells. This second assay revealed that transfected Alu and 7SL RNA can also cause suppression of HeLa cell proliferation. To investigate whether transcription or polymerase III (pol III) transcription factor binding was required for inhibitory activity, mutations were introduced into RNA pol III B block promoter elements in each of these genes. Suppression of [3H]thymidine incorporation was dependent on the presence of this pol III element in both genes; likewise, 7SL RNA-mediated growth suppression required the presence of the pol III B block promoter element. The results of this study indicate that Alu and 7SL RNA gene sequences interact with cellular factors that are important for HeLa cell proliferation and suggest that these pol III-transcribed elements may be involved in the regulation of cellular growth.

Transient analysis for antiproliferative gene activity.

A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.

A new vector using the human multidrug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells.

A new vector, pSK1.MDR, has been constructed for expressing nonselectable genes in eukaryotic cells. The vector uses the human multidrug resistance gene, MDR1, as a dominant selectable marker and contains an additional transcription unit plus a unique SalI cloning site for inserting nucleotide sequences to be expressed. To test this expression system, a cDNA (IL2R) for the 55-kDa interleukin-2 receptor was inserted into the SalI site, and the resulting plasmid was transfected into NIH3T3 cells. Cells which acquired the MDR1 gene were selected with colchicine, and cells with high levels of MDR1 expression were selected by growth in increasing concentrations of the drug. Drug resistant cells also expressed the cotransferred, nonselected IL2R gene, and its expression was increased to 740,000 receptors per cell by growing cells in high concentrations of colchicine. The MDR1 system represents a very efficient method for synthesizing large amounts of protein in a wide variety of eukaryotic cells.

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid.

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.

Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate.

To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.

Purification of transiently transfected cells by magnetic affinity cell sorting.

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

The 5'-flanking sequences of human globin genes contribute to tissue specific expression.

The specificity of expression of the isolated 5'-flanking sequences of the human beta- and epsilon-globin genes was examined in the K562 human erythroleukemia cell line, the murine erythroleukemia (MEL) cell, and in nonerythroid cell lines CV-1, HeLa-S3, and WI-38. Globin flanking sequences were active only in the erythroid K562 and MEL cell lines. Furthermore, the upstream sequence of the adult, beta-globin gene was active in MEL cells which express adult globin and not in K562 cells which express embryonic and fetal globins suggesting that tissue specificity resides in these upstream sequences. No expression was observed in K562 cells which have an embryonic-fetal hemoglobin phenotype.

A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system.

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.

Absence of renin-like activity in rat aorta and microvessels.

Vascular renin-like activity was studied in the aortas and the cerebral microvessels of Sprague-Dawley rats and in the aortas of spontaneously hypertensive rats. Methods were employed to maximize detection of tissue renin and to simultaneously minimize contamination of that activity by either plasma renin or nonspecific proteases capable of angiotensin I generation. To this end, renin activity was measured near its pH optimum; plasma renin was eliminated by nephrectomy; and nonspecific proteases such as cathepsin D were either inhibited by proteolytic blockers or removed by chromatography over immobilized bovine hemoglobin. Aortic vascular renin-like activity was detected in rats not subjected to nephrectomy and could be inhibited by preincubation of samples with antimouse renin antibody shown to cross-react and inhibit rat plasma renin activity. Furthermore, vascular renin-like activity disappeared after nephrectomy in parallel with the disappearance of plasma renin activity. In the absence of contaminating enzymatic activities, no tissue renin-like activity could be demonstrated in either aortas or cerebral microvessels of Sprague-Dawley rats or in aortas of spontaneously hypertensive rats.

Abundance and structure of globin mRNA in K562 human leukemia cells.

K562 cells can be reversibly induced by hemin to accumulate embryonic and fetal hemoglobins. The increase in individual globin mRNAs accompanying induction was measured by isolation and in vitro translation of poly A+ RNA from both control and induced cells. epsilon-Globin mRNA showed the greatest increase (threefold) with hemin induction, followed by zeta greater than gamma greater than alpha. These results were corroborated by measuring steady-state mRNA concentrations by a recently developed S1 nuclease mapping procedure. Changes in epsilon-, gamma-, and alpha-globin levels parallel the changes measured by in vitro translation. S1 nuclease mapping studies revealed that epsilon-, gamma-, and alpha-globin transcripts were correctly initiated and processed. In addition, correctly initiated and processed delta-globin transcripts were detected at low levels, increasing 1.5-fold following hemin induction. However, no beta-globin transcripts could be detected by this sensitive and specific assay. The beta-globin gene may be under developmental control in K562 cells, and this cell line may therefore provide a unique assay system for molecules involved in globin gene regulation.

Enzyme studies in tardive dyskinesia. III. Noradrenergic hyperactivity in a subgroup of dyskinetic patients.

This study was undertaken to test a hypothesis that tardive dyskinesia (TD) patients with high plasma dopamine-beta-hydroxylase (DBH) activity would also have other enzymatic alterations suggestive of noradrenergic hyperactivity. Plasma renin activity was chosen as a peripheral indicator of noradrenergic function. We measured plasma renin activity in three groups of female psychiatric inpatients over the age of 50: a TD group with high plasma DBH activity, a TD group with low plasma DBH activity, and a non-TD group. The high-DBH TD group had significantly greater plasma renin activity than the other two groups. Our results are consistent with the hypothesis that a subgroup of TD patients has relative noradrenergic hyperactivity.