Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.

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.

Chloramphenicol acetyltransferase as a reporter in Mammalian gene transfer.

Reporter genes can be used to advantage in a variety of different kinds of gene transfer experiments. One of the most common applications is the optimization of transfection methods. Owing to the large number of variables that influence the uptake and expression of exogenously introduced genes, it is extremely helpful to have available rapid, sensitive methods for determining transfection efficiency. A second frequent application of reporter genes is to study promoters and transcriptional enhancers. To rigorously map functional domains in transcriptional control elements it is often necessary to generate large numbers of deletions, insertions, and point mutations, and then carry out functional assays on each construct. As in optimization studies, an appropriate reporter gene can greatly facilitate the rate with which information is accumulated. Another gene regulation application involves measuring activity of transacting transcriptional regulatory proteins. In such experiments, one plasmid codes for the transacting factor, while the second cotransfected plasmid carries a target cis-acting element coupled to a reporter gene. Examples of more novel reporter gene uses include introducing translational stop codons into a reporter coding sequence to measure suppressor function in mammalian cells (1,2), and measuring transcriptional competence of UV-irradiated reporter plasmid DNA (3).

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.

Exposure of HeLa cells to 0(6)-alkylguanines increases sensitivity to the cytotoxic effects of alkylating agents.

Exposure of HeLa S3 cells to 0.4mM 0(6)-methylguanine or 0(6)-n-butylguanine for 24 h led to a substantial decrease in the activity of 0(6)-alkylguanine-DNA alkyltransferase. Such pretreatment caused a marked increase in the sensitivity of the cells to the cytotoxic effects of the cross-linking alkylating agent 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea and a smaller increase in the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. These results indicate that the repair of DNA by the alkyltransferase plays an important role in the protection of cells from the cytotoxic effects of certain alkylating agents particularly those such as 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea which ultimately lead to the formation of lethal interstrand cross-links.