By
From the Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115
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.
Considerable progress has been made in unraveling the
genetic elements of programmed cell death in lower
organisms such as Caenorhabditis elegans and Drosophila melanogaster (1, 2). Higher mammalian systems might possess
even more elaborate genetic pathways leading to the induction of apoptosis. However, since mammals are less amenable to genetic manipulation, our knowledge of their relevant genes might still be largely incomplete. Two genetic
approaches in mammalian cells have been described which facilitate the isolation of genes that inhibit apoptosis (3, 4).
Due to the transient apoptotic phenotype and the subsequent disappearance of the dead cells, the isolation of dominant genes that lead to the induction of apoptosis did not
seem practical. Nonetheless, here we report a simple genetic screen for dominant, apoptosis-inducing genes that is
based on easily scored morphological changes in apoptotic
cells. Using this screen, we have isolated a novel isoform of
the precursor protein for the protooncogene NDF (Neu
differentiation factor) which induces apoptosis on overexpression.
Isolation of Apoptosis-inducing Genes.
Kidney mRNA from 4-6wk-old FVB mice was normalized as described (5). The procedure uses the rapid association of abundant mRNA species with
their cDNAs covalently attached to latex beads which are subsequently removed by centrifugation. After two rounds of hybridization, 200 ng (of initially 2 µg) mRNA were retrieved and used
to generate a cDNA library with a cDNA synthesis kit (GIBCO
BRL, Gaithersburg, MD). After ligation of a BstXI adaptor (Invitrogen, San Diego, CA) and a NotI digest, the cDNAs were inserted into a modified pcDNA3 vector (Invitrogen) in which the neomycin resistance gene had been deleted. The DNA was electroporated into Escherichia coli SURE cells (Stratagene Corp., La
Jolla, CA) which were immediately frozen. Plating of aliquots revealed that the library contained ~1.1 × 105 independent clones.
Aliquots corresponding to 20 clones were thawed and grown up.
Miniprep DNA was isolated as described (6) and transfected into
293 cells in 24-well plates using the calcium phosphate co-precipitation method. After 18 h, the cells were inspected for apoptosis
induction with phase contrast microscopy. The bacteria pool whose
plasmid DNA caused morphological signs of apoptosis in 293 cells was spread on plates. Plasmid DNAs from individual bacteria
colonies were again transfected into 293 cells to isolate the active
clone.
Cell Transfections.
Plasmid DNAs were isolated with Qiagen
columns (Qiagen, Chatsworth, CA) and transfected into 293 or
baby hamster kidney (BHK) cells with the calcium phosphate coprecipitation method as described (7).
Expression Constructs.
The deletion mutants of Quantitative Apoptosis Assay.
BHK cells were transfected with
the respective plasmids and a DNA Isolation.
Low molecular weight DNA was isolated by
lysing the cells with 0.2% Triton X-100 on ice for 10 min. After
centrifugation, low molecular weight DNA was recovered in the
supernatant and phenol-extracted. The samples were loaded on a
2% agarose gel after RNA digestion.
Reverse Transcription PCR.
Total RNA was isolated (RNAstat;
Tel-Test Inc., Friendswood, TX) from the indicated tissues from
two 6-wk-old FVB mice. For the reverse transcriptase (RT)
PCR, 2 µg of total RNA were reverse transcribed with oligo dT
primers in a volume of 30 µl. 5 µl were used in a PCR reaction
with primers for
2b NDF were
created with PCR using suitable primers encompassing the
2b
NRG Kozak sequence, 200 ng template, and 20 cycles with Pwo
(Boehringer Mannheim, Indianapolis, IN) which has proofreading activity. All constructs were transcribed and translated in vitro
(TNT kit; Promega Corp., Madison, WI), and yielded products of
the expected sizes (not shown). In each case, two NDF expression constructs were generated by two independent PCR reactions.
-galactosidase (
-gal) expression
vector. When PCR-generated NDF deletion constructs were
used, two independently generated constructs were transfected in
two transfections each. 24 h later 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 (8).
-actin (Clontech, Palo Alto, CA), an oligo (5
AGCTTCTACAAAGCGGAG-3
) that spans the junction between the
and the 2 exon of
2b NDF (hybridizing to 8 bp of the 2 exon and 10 bp of the b exon) and a primer (5
-ATATCTAGATAAAGGCCAAGGGGTC-3
) that was complementary to
the
exon of
2b NDF. DNA templates for
-actin (~17 pg)
and
2b NDF (~200 pg) were used as positive controls, reverse
transcribed tRNA was used as a negative control. An initial denaturation time of 1 min and 25 cycles of 30 s at 94°C, 1 min at
52°C, and 1 min at 68°C were used in the PCR. The reaction
products were separated on 1% agarose gels and blots probed with
mouse
-actin or
2b NDF, respectively.
Reasoning that the machinery for apoptosis might already exist in latent form in dynamic organs (10), we used
normal mouse kidney as a RNA source for constructing an
expression library. To reduce the redundancy of the mRNA,
we normalized its sequence representation (5). From the
resulting, unamplified cDNA library, small aliquots were
grown up and plasmid miniprep DNAs were transfected into
the human kidney cell line 293 (Fig. 1). This cell line has the advantage of being highly transfectable and producing
an obvious apoptotic phenotype with membrane blebbing
and subsequent shrinkage of the cytoplasm. After screening
480 pools (~10% of the library) by phase contrast microscopy for apoptosis induction, a positive plasmid pool was
detected. A single apoptosis-inducing cDNA clone was isolated from this pool (Fig. 2). Transfection of as little as 200 ng
of this plasmid brought about apoptosis (not shown). Subsequent experiments demonstrated that this gene could also induce apoptosis in the hamster BHK cell line (see Fig. 4),
emphasizing the phylogenetic conservation of this function.
Isolation of low molecular weight DNA after transfecting
the expression plasmid into 293 cells revealed nucleosomal
DNA fragmentation, a biochemical hallmark of apoptosis
(11; Fig. 2 C).
The 2.7-kb insert cDNA was sequenced and was surprisingly identified as a member of the NDF family (12, 13).
NDF is a ligand for erbB receptor tyrosine kinases, some of
which have been implicated in a variety of human carcinomas (14). NDF gives rise to multiple alternative splice forms
which code for transmembrane precursor proteins from
which the secreted NDF molecules are proteolytically generated. The particular isoform described here contains the , 2, and b exons (see Fig. 4). This combination of exons
has not been previously described, establishing
2b NDF as
a novel isoform of NDF. No sequence resemblance to genes
known to be involved in apoptosis could be detected in the
database. Subsequently, RT-PCR was used to determine
the expression pattern of
2b NDF in mice (Fig. 3). The
results indicate that it is expressed at similar levels in most
tissues examined with higher amounts in brain and in stomach. Colon and muscle did not produce a signal in this experiment; however, extensive PCR amplification revealed low levels of expression in these organs as well (not shown).
Since the NDF precursor proteins are composed of multiple subdomains, we wished to delineate those domains
that are involved in apoptosis induction. Therefore, we engineered several deletion mutants of 2b NDF and scored
their ability to induce apoptosis after transient transfection
in BHK cells. Fig. 4 shows that deleting the immunoglobulin homology domain (IG) reduced apoptosis induction by
68%. Further deletions into the epidermal growth factor
homology domain abrogated the ability of
2b NDF to
cause cell death. However, mutant
2-198 could weakly
induce apoptosis in 293 cells which are more susceptible to
cell death induction (not shown). Overexpression of another mutant encompassing only the cytoplasmic domain
of
2b NDF was also inactive in 293 cells (not shown). Interestingly, the intracellular portion, which is not part of
the secreted moiety (15), was necessary for apoptosis induction (construct
263-460 in Fig. 4). The importance of
the intracellular domain of
2b NDF is also emphasized by
a mutant with a deletion of the b exon corresponding to
the recently described
2c NDF isoform (16). Its expression led to a 66% reduction in apoptosis. These data suggest
that NDF does not act as a secreted molecule in cell death induction (see below).
The supernatant from NDF-transfected 293 cells was able
to induce receptor tyrosine phosphorylation in MCF-7 cells
indicating that 293 cells can faithfully process 2b NDF
proteins (not shown). To further investigate whether a receptor-ligand interaction is involved in this apoptosis induction, we used a sensitive RT-PCR approach to detect
the presence of erbB receptors. In accordance with previous reports (9, 17), no expression of NDF-binding erbB genes
(erbB-3 and erbB-4) could be detected in 293 cells. However, erbB-2, which is unable to bind NDF (18), is expressed in 293 cells (Fig. 5 A). Stably transfecting erbB-2 and erbB-3 or transient co-transfection of a dominant-negative erbB-2
mutant (19) had no effect on
2b NDF's apoptosis induction (not shown). Furthermore, cells lying adjacent to NDFtransfected cells showed no signs of apoptosis (Fig. 5 B).
These results, the isolated appearance of apoptotic cells after transient transfection of 2b NDF (see Fig. 2 B), and
the observation that the supernatant from transfected 293 cells did not induce cell death in untransfected cells (not
shown) indicate that
2b NDF exerts its apoptotic effect
cell-autonomously and independently of known erbB receptors.
To integrate the apoptotic signal initiated by 2b NDF
into the cell death signal transduction pathway, we tried to
block cell death with known inhibitors of apoptosis. Both,
Bcl-2 and Bcl-x, the prototypical apoptosis repressors (20),
were capable of reducing cell death when co-transfected with
2b NDF (not shown). A 2.8-fold repression of apoptosis
was observed when baculovirus p35, an ICE protease inhibitor (21), was co-transfected indicating an activation of
ICE-cysteine proteases in NDF-mediated apoptosis (Fig. 5 C).
Interestingly, other inhibitors of ICE-like proteases, such as
the cowpox gene CrmA (22), was without any effect (Fig. 5 C), as were the tetrapeptide ZVAD-FMK (23) and Ich-1s
(24) (not shown).
In this study we have described the isolation of the novel
2b NDF splice variant using a genetic screen for dominant, apoptosis-inducing genes. The isolation of this gene
was unexpected. Though NDF was known to cause cellular responses of proliferation or growth arrest (13, 25), its
isolation as an inducer of apoptosis was surprising. Since the
2c isoform of NDF has been shown to function as an oncogene when overexpressed (16), it is worth noting that
several other oncogenes, most notably c-myc (26), participate in seemingly contradictory responses characterized by
proliferation or cell death. However, in contrast to these
oncogenes, NDF's apoptosis induction is separated from the
transformation process, since it can induce apoptosis in cells
which do not express NDF-binding receptors. The secreted
portion of NDF, which is identical between the
2c and
2b NDF isoforms, could therefore act as an oncoprotein by engaging erbB receptor tyrosine kinases in neighboring
cells, while the intracellular portion determines its apoptosis
induction. Both functions are activated when NDF is overexpressed, but apoptosis induction can kill the NDF-overproducing cell and might thereby protect the organism
against tumorigenesis. A functional role for the intracellular
domain of NDF has hitherto been unclear, but is suggested
by its evolutionary conservation (15). The different apoptotic activities of NDF splice variants in the intracellular domain might allow distinct expression levels in normal development without the induction of apoptosis.
Tumorigenesis by NDF overexpression would therefore require the inactivation of its apoptosis pathway. The genes in this signaling cascade are therefore good candidates for the "second hits" required for tumorigenesis which renders our findings an interesting subject for further studies.
Our assay system uses gene overexpression after transient transfection since most known apoptosis-inducing genes, like the ICE family of cysteine proteases, the "death domain" proteins and p53, achieve their effect at elevated concentrations. This might be the result of overcoming inhibitors and forming protein-protein interactions that stimulate the signal cascades leading to cell death. In any case, the power of our assay is demonstrated here by the identification of an unexpected and dominant property of the NDF precursor. We believe that this screen will uncover additional genes that lie at critical points in the genetically determined pathway that leads to programmed cell death.
Address correspondence to Philip Leder, Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, 200 Longwood Ave., Boston, MA 02115.
Received for publication 8 January 1997.
S. Grimm is supported by the AIDS-Stipendium of the Deutsche Krebsforschungszentrum, Heidelberg, Germany.We thank Drs. T. Lane, K. Fitzgerald, B.Z. Stanger, A. Elson, and I. Krane for helpful discussions. Thanks also to Drs. M. Kraus and B. Groner for providing expression plasmids for erbB receptors and to Drs. J. Yuan and L. Miller for sending plasmids for ICE inhibitors.
1. | Ellis, R.E., J. Yuan, and H.R. Horvitz. 1991. Mechanisms and functions of cell death. Ann. Rev. Cell Biol. 7: 663-698 . |
2. | Steller, H., and M.E. Grether. 1994. Programmed cell death in Drosophila. Neuron. 13: 1269-1274 [Medline]. |
3. | Vito, P., E. Lucana, and L. D'Adamio. 1996. Interfering with apoptosis: Ca2+-binding protein ALG-2 and Alzheimer's disease gene ALG-3. Science (Wash. DC). 271: 521-525 [Abstract]. |
4. | Jaattela, M., M. Benedict, M. Tewari, J.A. Shayman, and V.M. Dixit. 1995. Bcl-x and Bcl-2 inhibit TNF and Fas- induced apoptosis and activation of phospholipase A2 in breast carcinoma cells. Oncogene. 10: 2297-2305 [Medline]. |
5. | Sasaki, Y.F., D. Ayusawa, and M. Oishi. 1994. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system. Nucleic Acids Res. 22: 987-992 [Abstract]. |
6. | Feliciello, I., and G. Chinali. 1993. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from E. coli. Anal. Biochem. 212: 394-401 [Medline]. |
7. | Roussel, M.S., C.W. Rettenmeier, A.T. Look, and C.J. Scherr. 1984. Cell surface expression of v-fms coded glycoprotein is required for transformation. Mol. Cell Biol. 4: 1999-2009 [Medline]. |
8. | Stanger, B.Z., P. Leder, T.-H. Lee, E. Kim, and B. Seed. 1995. RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell. 81: 513-523 [Medline]. |
9. |
Chan, S.D.H.,
D.M. Antoniucci,
K.S. Fok,
M.L. Alajoki,
R.N. Harkins, and
S.G. Wada.
1995.
Heregulin activation of
extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.
J. Biol. Chem.
270:
22608-22613
|
10. | Weil, M., M.D. Jacobson, H.S.R. Coles, T.J. Davis, R.L. Gardner, K.D. Raff, and M.C. Raff. 1996. Constitutive expression of the machinery for programmed cell death. J. Cell Biol. 133: 1053-1059 [Abstract]. |
11. | Wyllie, A.H.. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (Lond.). 284: 555-556 [Medline]. |
12. | Wen, D., E. Peles, R. Cupples, S.V. Suggs, S.S. Bacus, Y. Luo, G. Trail, S. Hu, S.M. Silbiger, R. Ben-Levy, et al . 1992. Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell. 69: 559-572 [Medline]. |
13. | Holmes, W.E., M.X. Sliwkowski, R.W. Akita, W.J. Henzel, J. Lee, J.W. Park, D. Yansura, N. Abadi, H. Raab, G.D. Lewis, et al . 1992. Identification of heregulin, a specific activator of p185 erbB2. Science (Wash. DC). 256: 1205-1210 [Medline]. |
14. | Prigent, S.A., and N.R. Lemoine. 1992. The type 1 (EGFRrelated) family of growth factors receptors and their ligands. Prog. Growth Factor Res. 4: 1-24 [Medline]. |
15. | Wen, D., S.V. Suggs, D. Karunagaran, N. Liu, R.L. Cupples, Y. Luo, A.M. Janssen, N. Ben-Baruch, D.B. Trollinger, V.L. Jacobson, et al . 1994. Structural and functional aspects of the multiplicity of Neu differentiation factors. Mol. Cell Biol. 14: 1909-1919 [Abstract]. |
16. | Krane, I.M., and P. Leder. 1996. NDF/heregulin induced persistence of terminal end buds and adenocarcinomas in the mammary of transgenic mice. Oncogene. 12: 1781-1789 [Medline]. |
17. | Levkowitz, G., L.N. Klapper, E. Tzahar, A. Freywals, M. Sela, and Y. Yarden. 1996. Coupling of the c-Cbl protooncogene product to Erb-1/EGF-receptor but not to other ErbB proteins. Oncogene. 12: 1117-1125 [Medline]. |
18. | Plowman, G.D., I.M. Green, J.-M. Culouscon, G.W. Rothwell, and B. Sharon. 1993. Heregulin induces tyrosine phosphorylation of HER4/p180 erbB4. Nature (Lond.). 366: 473-475 [Medline]. |
19. | Messerle, K., I. Schlegel, N. Hynes, and B. Groner. 1994. NIH/ 3T3 cells transformed with the activated erbB-2 oncogene can be phenotypically reverted by a kinase deficient, dominant negative erbB-2. Mol. Cell. Endocrinol. 105: 1-10 [Medline]. |
20. | Korsmeyer, S.J.. 1995. Regulators of cell death. Trends Genet. 11: 101-105 [Medline]. |
21. | Clem, R.I., and L.K. Miller. 1994. Control of programmed cell death by the baculovirus genes p35 and iap. Mol. Cell Biol. 14: 5212-5222 [Abstract]. |
22. | Miura, M., H. Zhu, R. Rotello, E.A. Hartwig, and J. Yuan. 1993. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homologue of the C. elegans cell death gene ced-3. Cell. 75: 653-660 [Medline]. |
23. | Fearnhead, H.O., D. Dinsdale, and G.M. Cohen. 1995. An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes. FEBS Lett. 375: 283-288 [Medline]. |
24. | Wang, L., M. Miura, L. Bergeron, H. Zhu, and J. Yuan. 1994. Ich-1, an ICE/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell. 78: 739-750 [Medline]. |
25. | Peles, E., S.S. Bacus, R.A. Koski, H.S. Lu, D. Wen, S.G. Odgen, R. Ben-Levy, and Y. Yarden. 1992. Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell. 69: 205-216 [Medline]. |
26. | Evan, G.I., A.H. Wyllie, C.S. Gilbert, T.D. Littlewood, H. Land, M. Brooks, C.M. Waters, L.Z. Penn, and D.C. Hancock. 1992. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 69: 119-128 [Medline]. |