Embryonic inducers, growth factors, transcription factors and oncogenes

Cell Differentiation and Development, Elsevier Scientific Publishers Ireland,

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26 (1989) 163-171 Ltd.

CDF 00583

Review

Embryonic inducers, growth factors, transcription factors and oncogenes , Walter Knijchel and Heinz Tiedemann Institut ftir Molekularbiologie

und Biochemie, Freie Universitiir Berlin, Berlin, F.R. G. (Received

4 January

A great challenge to all scientists working in the fields of molecular biology is to understand the molecular basis of normal growth and differentiation and that of uncontrolled growth of cancer cells. Research on malignant cell transformation has been greatly stimulated after the discovery of viral and cellular oncogenes. The recent discoveries that certain growth factors are active in embryonic tissue induction and that previously described inducing factors are probably related to these protein families, have raised the question whether cellular oncogenes are also involved in the embryonic differentiation process. This assumption is based on the observation that certain oncogenes are essentially homologous to some growth factors or receptor proteins.

1989)

the viral genomes. However, some oncogenes were also discovered in DNA tumor viruses and in chemically induced as well as in spontaneous tumors. There are several mechanisms which lead to the generation of oncogenes from proto-oncogenes. Point mutations The ras oncogene was detected when DNA from a human bladder tumor was transferred into normal mouse fibroblasts (Krontiris and Cooper, 1981; Murray et al., 1981). It is also present in the Harvey strain of the murine sarcoma virus. The difference between the normal ras proto-oncogene and the ras oncogene, active in tumors, is manifested in only one nucleotide mutation leading to an amino acid exchange (Reddy et al., 1982; Tabin et al., 1982; Taparowsky et al., 1982).

Origin of oncogenes Many oncogenes have been discovered in transforming RNA tumor viruses (retroviruses). Apparently the viral oncogenes (v-oncogenes) arose from cellular genes (c-oncogenes = proto-oncogenes) that are normal genes which were captured by the viruses and integrated in mutated form in

Correspondence address: W. Knijchel, Institut fiir Molekularbiologie und Biochemie, Freie Universitlt Berlin, Amimallee 22, 1000 Berlin 33, F.R.G. 0922-3371/89/$03.50

0 1989 Elsevier Scientific

Publishers

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Chromosomal rearrangements Chromosomal rearrangements, i.e., gene translocations, inversions or deletions can also lead to the generation of oncogenes. Various types of rearrangements have been observed in case of the myc gene (reviewed by Fahrlander and Marcu, 1986). Gene amplifications Amplification of the N-myc has been found in neuroblastomas (Schwab et al., 1983) and of L-myc in small cell lung tumors (Nau et al., 1985). Ltd.

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Relationship between oncogenes and growth factors

During recent years it turned out that, on the basis of sequence homologies, many oncogene products are structurally related to growth factors families (reviewed by Kahn and Graf, 1986). A clear relationship between oncogenes, protooncogenes and growth factors controlling cell proliferation has meanwhile been found in numerous cases. The following examples are by no means comprehensive and present only part of a rapidly growing list. The sis proto-oncogene encodes the PDGF /3 chain (Doolittle et al., 1983; Waterfield et al., 1983) (PDGF = platelet derived growth factor is a dimer of the 17 kDa (Ychain and the 16 kDa /I chain). The proto-oncogene erb B encodes a truncated EGF (epidermal growth factor) receptor (Downward et al., 1984), which, in turn, is related to TGF-cy (transforming growth factor) (Marquardt et al., 1984). The proto-oncogene erb A encodes a thyroid hormone receptor (Weinberger et al., 1986) being structurally related to the steroid hormone receptors, c-fm represents the receptor protein for CSFl (CSF = colony stimulating factor) (Sherr et al., 1985). The int 2 protooncogene is related to aFGF and bFGF (acidic and basic fibroblast growth factors) (Dickson and Peters, 1987). These FGFs are also related to FGF 5, an oncogene, which was isolated from a human bladder carcinoma (Zhan et al., 1988) and to the hst oncogene, which was initially identified by transfection of NIH 3T3 cells with DNA from several cancer and non-cancer tissues (Taira et al., 1987; Yoshida et al., 1987) and is probably identical to the oncogene, found in Kaposi sarcomas (Delli Bovi et al., 1987). The ras oncogene product seems to be related to the G protein family (for review see Masters and Bourne, 1986). It has a GTP binding activity and is probably involved in cAMP/cGMP second messenger dependent signal transduction. The ras proto-oncogene can further induce production of TGFs (for review see Sporn and Roberts, 1985). The TGF-/_I family comprises, besides TGF-/31 and TGF-P2, the Mtillerian inhibiting substance (Cate et al., 1986), the product from the Drosophila melanogaster decapentaplegic gene complex (DPP-C) (Padgett et al., 1987), p-

subunits of inhibin (Mason et al., 1985), the erythroid differentiation factor, which is completely identical to the & subunit of inhibin (Murata et al., 1988), and the Vgl gene transcripts, which are localized in the vegetal hemisphere of Xenopus eggs (Weeks and Melton, 1987). TGF-/3 was first identified by its ability to cause phenotypic transformation of rat fibroblasts (Roberts et al., 1981) and embryo-derived AKR-2B cells (Moses et al., 1981), but has subsequently been shown to be similar, if not identical, to a previously identified mitosis inhibitor in African green monkey (BSC-1) cells (Holley et al., 1978; Tucker et al., 1984). From the initially confusing variety of positive and negative effects on cell proliferation - depending on cells and other factors present - emerged the picture of positive and negative signals in growth control, which are reflected by autocrine secretion and by positive as well as negative feedback loops (for reviews see Spom and Roberts, 1985, 1988; Heldin et al., 1987). For a long time it was supposed that protooncogenes and growth factors are involved in cell differentiation, and only recently it could be shown that substances related to growth factors participate in the determination of certain pathways of the differentiation of totipotent cells. A great number of growth factors and proto-oncogenes is differentially expressed in embryonic tissues (for review see Mercola and Stiles, 1988), and there are accumulating data that they serve important functions in embryogenesis. Moreover, from recent experiments it turned out that certain members of growth factor families are directly involved in developmental processes. This demonstrates that members of certain growth factor families do not show only mitogenic but also morphogenic effects. The gene encoding the epidermal growth factor shares partial homologies with the homeotic loci lin-12 in C. elegans and Notch in Drosophila, the latter preventing the differentiation process of ectodermal cells to neural cells (Greenwald, 1985; Wharton et al., 1985; Knust et al., 1987). The sequence of the Drosophila segment polarity gene wingless corresponds to the mammalian protooncogene int-1 (Rijsewijk et al., 1987). Another example is the relation of the Drosophila em-

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bryonic polarity gene dorsal to the vertebrate proto-oncogene rel (Steward, 1987). Different members of the FGF and TGF-p families play important roles in embryonic differentiation. Besides the erythroid differentiation factor and the Miillerian inhibiting substance, which leads to a regression of the Miillerian duct in male embryos, the decapentaplegic gene product contributes to the determination of dorsal structures in Drosophila embryos and later to the morphogenesis of the imaginal disks. It is also expressed in mesoderm and endoderm derived tissues. Perhaps it is a common principle that genes or related genes which are active in early embryos are reactivated at later stages of development and that the gene products serve different functions. This may reflect a modulation of gene activity in the context of different gene networks.

Embryonic inducers In the past, amphibia have been proven for several reasons to be a very suitable model-system to study vertebrate embryogenesis. The search for chemical substances with inducing activity began in the thirties, but soon led to results which were difficult to interpret. It was not until the fifties that mesoderm-inducing extracts were obtained from guinea pig bone marrow (Yamada, 1958, 1961), which preferentially induces trunk and tail structures (Toivonen, 1953), and from 9-11-day-old chicken embryos (Tiedemann and Tiedemann, 1956). From the latter extract a protein could be highly purified which induces mesoderm- and endoderm-derived tissues (Born et al., 1985) and in turn leads to a change of cell affinities (Kocher-Becker et al., 1965). Since this factor in vivo is in part bound to acidic proteoglycans and in vitro binds to heparin-sepharose (Born et al., 1987), it was tempting to investigate whether heparin binding growth factors of the FGF protein family also exhibit a mesoderm-inducing activity. Indeed, several laboratories have demonstrated that aFGF as well as bFGF can induce the formation of mesodermal tissues when applied to ectoderm explants isolated from late blastulae/ early gastrulae of Xenopus laeois (Kimelman and

Kirschner, 1987; Knijchel et al., 1987; Slack et al., 1987; Grunz et al., 1988). Like the chicken-derived factor, both FGFs induce at high concentrations preferentially muscle and at low concentrations endothelia lined vesicles which contain, besides some pycnotic cells, single cells with the typical appearance of immature blood cells. However, in contrast to Xenopus ectoderm, explants from Triturus alpestris differentiated in addition to neural tissue. Whether the neural tissue is induced by the primarily induced mesoderm (as in normal development) is not yet known. These explants showed a very large number of mitoses (Km&he1 et al., in preparation). Despite the similarities in heparin binding and some other chemical properties, the chicken-derived factor and the FGFs differ in several properties such as hydrophobicity or inactivation by reduction of disulfide bridges. In these properties the chicken factor is more closely related to TGFp, and both factors can efficiently be extracted from tissues by acidic ethanol. TGF-j? comprises at least two different factors, TGF-/?l and TGFp2, which show an amino acid sequence homology of approximately 70%. TGFj32 induces mesodermal tissues (muscle) in Xenopus Iueuis ectoderm explants, whereas TGF-bl does not (Rosa et al., 1988). In ectoderm explants of Triturus alpestris, TGF-fil as well as TGF-/32 induce endothelium-lined vesicles, mesenchyme, and, in elongated explants, a blastema tissue. Metameric strands of cuboidal cells originate from this growth center. In some explants large masses of endothelial tissue are arranged in a capillary-like network (Kniichel et al., 1987, 1989). TGF-P2 induces, in addition, muscle and notochord. The reason for the different susceptibility of Triturus and Xenopus ectoderm to TGF-Pl is not yet known, perhaps it is due to different numbers of receptors. Although the chicken factor is most likely not identical with the FGFs or TGFs, it is presumably related to one of these growth factor families. This also holds true for the recently described factor from a transformed Xenopw fibroblast cell line, the XTC factor (Smith, 1987; Smith et al., 1988; Grunz et al., 1989). This factor shows a cross-reaction to antibodies against TGF82 (Rosa et al., 1988), but since the amino acid

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sequence has not yet been determined, the exact relationship still remains unknown. In any case, the growth factors have to be applied in much higher concentrations than the chicken factor and the XTC factor to achieve a comparative level of mesodermal induction in isolated ectoderm explants, making it unlikely that they are the best candidates for natural inducing factors. It has to be mentioned, however, that transcripts of FGF and TGF-8 related sequences have been found in the egg and early embryo (Kirnelman and Kirschner, 1987; Weeks and Melton, 1987). Whether the corresponding proteins also exhibit an inducing activity remains to be elucidated. Another question arises from the observation that mesodermal induction can be achieved with such different factors as FGF and TGF which share no amino acid sequence homologies. This may be explained by the hypothesis that these factors interfere with different targets in signal transduction (either from cell to cell or from the plasma membrane to the nucleus). It is also reasonable to assume that there is a whole network of factors and that TGF-j3 and FGF like molecules interfere with this network at different levels of hierarchy. In this context it is interesting to note that TGF-/3 induces the synthesis of interleukin 1 (Wahl et al., 1987), a factor closely related to FGF. Moreover, medium conditioned with TGFP-treated ectoderm exhibits a higher inducing activity than TGF-P-conditioned medium alone (Knijchel et al., 1989). This makes it likely that secondary factors are secreted into the medium and that their synthesis is triggered by activating the induction process. A neuralizing factor, which is protein or glycoprotein in nature, is released from the dorsal mesoderm (John et al., 1983) and acts on the surface of the plasma membrane of the competent ectoderm cells (Born et al., 1986). This factor has been partially purified from gastrula/neurula staged Xen0pu.s embryos and shown to increase the activity of protein kinases (Davids, 1988). This corresponds to the observations that phorbol 12myristate 13-acetate (PMA or TPA = tumorpromoting agent) stimulates neural differentiation and mitosis in Triturus alpestris and Xenopus laevis ectoderm (Davids et al., 1987; Otte et al., 1988),

and it implies that in neural induction either protein kinase C or related Ca2+ independent enzymes are involved. The mechanism of their activation is, however, not yet known.

Gene activation in induced tissues

Whatever may be identified as the primary agent to initiate the induction process, the apparent result is the activation of special genes or whole cascades of genes finally leading to the phenotypic appearance of differentiated cells. Accordingly, it is conceivable to postulate a model up to which primary factors are bound to extracellular or intracellular receptors, and then, with or without signal transduction, a signal is passed to the nucleus, the result of which is transcriptional activation. The corresponding gene product, which might present a secondary factor, could activate a whole cascade of genes either in the same cell or in neighboring cells. Interestingly, a critical mass of cells is required to induce embryonic differentiation (Grunz, 1979; Minuth and Grunz, 1980; Gurdon, 1988). The transcription process itself requires transcription factors, which are necessary to form a stable transcription complex. Transcription factors, which have been shown to play important roles in embryogenesis, can mainly be classified into two groups, the helix turn helix proteins containing the homeobox and the zinc finger proteins (for reviews see Gehring and Hiromi, 1986; Klug and Rhodes, 1987; Dressler and Gruss, 1988; Evans and Hollenberg, 1988). Associated with these DNA binding motifs different conserved structural modules have been identified, which allow the definition of further potentially overlapping subgroups, i.e., the paired box (Bopp et al., 1986), the POU domain (reviewed by Robertson, 1988) and the finger-associated boxes (Knochel et al., in preparation). Pioneering work with Drosophila, which was greatly facilitated by the availability of numerous mutants, led to the discovery of the general importance of these molecules in body plan formation (reviewed in Scott and O’Farrel, 1986; Gaul and J&Me, 1987). Using the cloned genes as hybridization probes, corresponding genes have meanwhile been discovered

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in a wide variety of eukaryotic cells including mammalian tissues. In a few cases, the biological function of these genes as developmental regulators in vertebrates has already been demonstrated. For example, a homeotic gene product is involved in frog somite formation (Harvey and Melton, 1988), a paired box containing mouse gene has a causative role in the generation of the vertebral column (Balling et al., 1988), and the retinoic acid receptor is a putative zinc finger protein, which is related to the receptors of steroid hormones and thyroid hormones (Petkovich et al., 1987). Retinoic acid is supposed to be a natural morphogen providing positional information in the polarity of chicken limb bud formation (Thaller and Eichele, 1987). Even if we cannot decide at the moment whether all members of these protein families really serve as transcription factors, it is reasonable to assume that a major portion of them can be aligned for this function. Especially the finger protein family comprises several hundred members (Koster et al., 1988; Nietfeld et al., 1989), part of which may have a general transcriptionactivating function without stage or tissue specificity, but another part is stage specifically transcribed during Xenopus embryogenesis and may activate the transcription of few or only one target gene. What is the cormecting link between transcription factors and growth factors? There is accumulating evidence that specific transcription factors are synthesized upon the administration of various growth factors. For example, myc and fos proto-oncogene products which are synthesized as a cellular response upon addition of mitogens do likely act as transcription factors, at least their expression is found in the nucleus after stimulation of cultured cells with growth factors (Kelly et al., 1983; Greenberg and Ziff, 1984; Kruijer et al., 1984; Mtiller et al., 1984). The nerve growth factor (NGF) induces NGFI-A, a possible transcriptional regulatory factor (Milbrandt, 1987). Stimulation of resting cells with growth factors leads to expression of specific finger proteins (Chavrier et al., 1988; Christy et al., 1988; Joseph et al., 1988; Lemaire et al., 1988; Sukbatme et al., 1988) TGF-P promotes the synthesis of matrix proteins and proteoglycans (Chen et al., 1987) probably by

transcription activation of the corresponding genes. It could be shown that TGF-fi induces the expression of a type I collagen gene mediated by a binding site for the transcription factor NFl (Rossi et al., 1988). On the other hand, it is interesting to note that there is also a close relationship between transcription activators and oncogene products. The products of myc and f0.r oncogenes have already been mentioned, as well as the identity between the erb A and the thyroid hormone receptor and its homology to other zinc finger hormone receptors. The fos-associated protein p39 is the product of the jun proto-oncogene (Rauscher et al., 1988), and it encodes a DNA-binding protein with structural and functional properties of transcription factor AP-1 (Bohmann et al., 1987). This finding also shows that there is a DNA as well as a protein binding activity associated with nuclear oncogene products, similar to that already reported for the well known transcription complex on oocyte-specific 5 S RNA genes, in which the zinc finger protein TFIIIA only in combination with other transcription factors leads to the formation of a stable and functional transcriptional complex (Wolffe and Brown, 1988).

Concluding remarks

The various data which have been reviewed in this article render convincing evidence that there is a close relationship between growth factors, embryonic inducers and oncogene products. These molecules themselves act on the expression of transcription activators, which again are related to other oncogenes. This suggests that the malignant potential of oncogenes is mainly due to the fact that the cellular genes, which have been captured by the viruses, serve in extremely important functions in cell differentiation as regulatory molecules (it is rather unlikely that a globin or actin gene will do this function). Thus it can be anticipated that cellular counterparts for many v-oncogenes will be found in form of regulatory molecules. Vice versa, only those cellular genes are good candidates for oncogenes which are involved in the regulation of cell cycle and cell differentiation processes.

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