An apoptosis-inducing isoform of neu differentiation factor (NDF) identified using a novel screen for dominant, apoptosis-inducing genes - PubMed (original) (raw)

An apoptosis-inducing isoform of neu differentiation factor (NDF) identified using a novel screen for dominant, apoptosis-inducing genes

S Grimm et al. J Exp Med. 1997.

Free PMC article

Abstract

Apoptosis is a genetically programmed series of events that results in cell death. As a consequence, it is difficult to identify dominant genes that play a role in this process using genetic selections in conventional cell culture systems. Accordingly, we have established an efficient expression screen to isolate dominant, apoptosis-inducing genes. The assay is based on the apoptotic morphology induced in the human kidney cell line 293 after transient transfection of small plasmid pools from normalized cDNA expression libraries. Using this assay, we isolated a novel isoform of the proto-oncogene Neu differentiation factor (NDF), a ligand for erbB receptor tyrosine kinases. Several lines of experimental evidence indicate that this gene kills in a cell-autonomous fashion and independently of known erbB receptors. This apoptotic property of an NDF isoform is readily contrasted with NDF's transforming potential and might balance the tendency to tumorigenesis in cells that overexpress NDF.

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Figures

Figure 1

Figure 1

Outline of the assay for cloning apoptosis-inducing genes. After normalization of the mRNA, a library was constructed in a mammalian expression vector. DNAs from pools of ∼20 bacteria clones were transiently transfected into 293 cells. The bacteria of the pool which induced morphological signs of apoptosis in cells (as indicated in the figure by a cell with altered shape) were plated and individual clone DNAs were once more transiently transfected. The positive clone was again identified by inspecting the cells for apoptosis induction.

Figure 2

Figure 2

Apoptosis induction by the isolated expression plasmid. 293 cells were transiently transfected with the empty vector (A) or the isolated plasmid clone (B). After 12 h, incubation phase contrast pictures at a 320-fold magnification were taken. (C) Expression of the isolated clone leads to DNA degradation. 293 cells were transfected with the vector (lane 1) or with the expression plasmid DNA (lane 2). After 16 h, low molecular weight DNA was isolated, separated on a 2% agarose gel, and stained with ethidium bromide.

Figure 4

Figure 4

Mapping of the apoptosis-inducing domains in β2b NDF with deletion mutants in BHK cells. The various domains are indicated on top of the diagrammatic representation of wild-type β2b NDF. IG, immunoglobulin domain; Glyco, glycosylated domain; EGF, epidermal growth factor homology domain; β, sequences of β exon; 2, sequences from exon 2; TM, transmembrane domain; Cyto, cytoplasmatic domain; b, sequences from exon b. The names of the deletion mutants indicate the deleted amino acids. Wild type or the deletion mutants of β2b NDF (4 μg) were transfected into BHK cells together with a marker plasmid (1 μg) for β-gal staining. 24 h after transfection the cells were stained with X-gal and the extent of apoptosis was measured by counting morphologically apoptotic, blue cells and determining their percentage of the total number of blue, transfected cells. Shown are the means and the standard deviation of a total of at least 1,000 counted cells from four independent experiments.

Figure 3

Figure 3

Expression profile of β2b NDF in mice as measured by RTPCR. β2b NDF-specific primers were used to amplify a 724-bp long DNA. The efficiency of the RT reaction was controlled by co-amplifying a 540-bp long β-actin fragment. Both products were detected by Southern blotting. The β2b NDF blot was exposed for 12 h, and the β-actin blot for 2 h.

Figure 5

Figure 5

Characterization of the apoptotic signal generated by β2b NDF. (A) Absence of NDF-binding erbB receptors in 293 cells as detected by RT-PCR. RNAs of the human mammary cell line MCF-7 and 293 cells were amplified, with or without earlier treatment with RT, with specific primers for the indicated genes. The PCR products were separated in agarose gels, blotted onto filters, and hybridized with gene-specific probes. β-actin was amplified to demonstrate the efficiency of the RT reactions. (B) Cells adjacent to NDF-transfected cells do not undergo apoptosis. BHK cells were transfected with the empty vector or β2b NDF (4 μg each) together with the marker plasmid for β-gal (1 μg), and apoptosis was monitored as in Fig. 4. Apoptosis was also measured in cells surrounding NDF-transfected, nonapoptotic (a) and apoptotic (b) cells. (C) β2b NDF-induced apoptosis is inhibitable by the ICE proteases inhibitor p35. BHK cells were transfected with β2b NDF (2 μg) and vector, CrmA, or p35 expression plasmids (2 μg). Apoptosis induction was measured as in Fig. 4.

Figure 5

Figure 5

Characterization of the apoptotic signal generated by β2b NDF. (A) Absence of NDF-binding erbB receptors in 293 cells as detected by RT-PCR. RNAs of the human mammary cell line MCF-7 and 293 cells were amplified, with or without earlier treatment with RT, with specific primers for the indicated genes. The PCR products were separated in agarose gels, blotted onto filters, and hybridized with gene-specific probes. β-actin was amplified to demonstrate the efficiency of the RT reactions. (B) Cells adjacent to NDF-transfected cells do not undergo apoptosis. BHK cells were transfected with the empty vector or β2b NDF (4 μg each) together with the marker plasmid for β-gal (1 μg), and apoptosis was monitored as in Fig. 4. Apoptosis was also measured in cells surrounding NDF-transfected, nonapoptotic (a) and apoptotic (b) cells. (C) β2b NDF-induced apoptosis is inhibitable by the ICE proteases inhibitor p35. BHK cells were transfected with β2b NDF (2 μg) and vector, CrmA, or p35 expression plasmids (2 μg). Apoptosis induction was measured as in Fig. 4.

Figure 5

Figure 5

Characterization of the apoptotic signal generated by β2b NDF. (A) Absence of NDF-binding erbB receptors in 293 cells as detected by RT-PCR. RNAs of the human mammary cell line MCF-7 and 293 cells were amplified, with or without earlier treatment with RT, with specific primers for the indicated genes. The PCR products were separated in agarose gels, blotted onto filters, and hybridized with gene-specific probes. β-actin was amplified to demonstrate the efficiency of the RT reactions. (B) Cells adjacent to NDF-transfected cells do not undergo apoptosis. BHK cells were transfected with the empty vector or β2b NDF (4 μg each) together with the marker plasmid for β-gal (1 μg), and apoptosis was monitored as in Fig. 4. Apoptosis was also measured in cells surrounding NDF-transfected, nonapoptotic (a) and apoptotic (b) cells. (C) β2b NDF-induced apoptosis is inhibitable by the ICE proteases inhibitor p35. BHK cells were transfected with β2b NDF (2 μg) and vector, CrmA, or p35 expression plasmids (2 μg). Apoptosis induction was measured as in Fig. 4.

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