Apoptosis induction in NDF-caused tumors. Hematoxylin and eosin–stained tissues from an adenocarcinoma in the mammary gland of NDF-transgenic mice, WT mammary gland, and a myc-induced tumor were probed with the TUNEL technique that detects DNA ends that are generated during apoptosis.

Model for the apoptotic and tumorigenic activity of NDF. A cell that overexpresses the NDF-precursor releases high amounts of the secreted NDF form. Its interaction with erbB receptors can lead to the subsequent proliferation of neighboring cells and might constitute the first step towards transformation. NDF's apoptosis-inducing activity is concomitantly activated when NDF is overexpressed and thereby kills this potentially dangerous cell.

Apoptosis induction by different NDF isoforms. Expression plasmids (4 μg) of the indicated NDF isoforms or of TGF-α were transfected together with a β-gal vector (1 μg) into BHK cells. Apoptosis induction was measured as in Fig. 1. The structure of each NDF isoform is schematically depicted in the diagram. Each subdomain is named as in Fig. 1. α and a denote the α or the a exon in the extra- or intracellular domain, respectively.

Apoptosis induction of NDF deletion mutants with and without a signal sequence. The domains of wild-type β2b NDF are indicated on top of the diagrammatic representation. IG, immunoglobulin homology domain; Glyco, glycosylated domain; EGF, epidermal growth factor domain; β, sequences of β exon; 2, sequences of exon 2; TM, transmembrane domain; Cyto, cytoplasmic domain; b, sequences of b exon; Sig, signal sequence of erbB-3. The names of the deletion constructs denote the deleted amino acids of wild-type β2b NDF. As a control in the case of the constructs that did not induce apoptosis (Δ 2-262, Sig Δ 2-262, and Δ 2-198), myc-tagged versions were transfected into BHK cells and the protein products were analyzed on SDS gels and detected with anti-myc-tag antibody. In each case, protein of appropriate mobility was detected (data not shown). 1.5 μg of each expression construct were transfected into BHK cells with 1 μg of a β-gal expression vector. 24 h after transfection the cells were stained for β-gal activity and the percentage of apoptotic and blue cells of all blue cells was determined using morphological inspection. Shown are the means and SD of at least 1,000 counted cells of four independent transfections.