A network of p73, p53 and Egr1 is required for efficient apoptosis in tumor cells.

p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.

The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling.

The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.

Method for cloning in vivo targets of the Egr-1 transcription factor.

A methodology is described that allows the in vivo trapping of transcription factors to their target regulatory elements in multiple genes simultaneously. Cross-linking using formaldehyde is the first of several steps to isolate, purify, clone and characterize multiple gene promoter DNA fragments. The example that we use indicates that the TGF beta 1 gene is a direct target induced by Egr-1 in HT1080 cells that express constitutive Egr-1, thus explaining the growth retardation that follows Egr-1 expression. The genes identified using this procedure reflect the specific activities of Egr-1 at that moment in the cell and provide a method for confirmation of genes that are the direct targets of Egr-1 action.

Shigella actin-based motility in the presence of truncated vinculin.

Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility.

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis.

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

Preneoplastic mammary tumor markers: Cripto and Amphiregulin are overexpressed in hyperplastic stages of tumor progression in transgenic mice.

Amphiregulin (Ar) and Cripto (Cr) are autocrine growth factors for mammary cells and both have been observed to exhibit high expression in human mammary tumors, in contrast with adjacent tissues. To investigate whether Ar and Cr play roles in the progression of mammary cell proliferation to unregulated growth and tumor formation, the levels of expression were examined in transgenic mice (TGM) that over-express several different oncogenes: MMTV-Polyoma virus middle T antigen (MMTV-PyMT), MMTV-c-ErbB2 (c-neu, HER2) and MT-hTGF alpha. These transgenic mice all produce mammary tumors but with different rates of progression. The levels of Ar were induced up to 10-fold in association with hyperplasia in 2 of the TGM. Cr overexpression was consistently observed in hyperplastic mammary glands in all the animal models, decreasing in overt tumors in 2 of the TGM models. In MMTV-PyMT mammary glands, the levels of Cr expression rose 7- to 10-fold in hyperplastic tissue and 25-fold the levels in tumors compared to age-matched transgene negative mice. Ar and especially Cr thus should have potential value as markers of preneoplastic change in mammary tissue.

Abrogation of the Cripto gene in mouse leads to failure of postgastrulation morphogenesis and lack of differentiation of cardiomyocytes.

Cripto-1(Cr1) protein encoded by the tdgf1 gene, is a secreted growth factor that is expressed early in embryonic development and is re-expressed in some tumors of the breast and colon. During embryonic development, Cr1 is expressed in inner cell mass cells and the primitive streak, and later is restricted to the developing heart. To investigate the role of Cr1 during mouse development, mice were generated that contain a null mutation of both Cr1 genes, derived from homologous recombination in embryonic stem cells. No homozygous Cr1-/- mice were born, indicating that Cr1 is necessary for embryonic development. Embryos initiated gastrulation and some embryos produced mesoderm up to day E7.5. Increasingly aberrant morphogenesis gave rise to disordered neuroepithelium that failed to produce a recognizable neural tube, or head-fold. Although some biochemical markers of differentiating ectoderm, mesoderm and endoderm were expressed, all the cardiac-specific markers were absent from day E8.7 embryos: (&agr;)MHC, betaMHC, MLC2A, MLC2V and ANF, whereas they were expressed in wild-type embryos. The yolk sac and placental tissues continued development in the absence of the embryo until day E9.5 but lacked large yolk sac blood vessels. Chimeric mice were constructed by microinjection of double targeted Cr1(-/- )embryonic stem cells into normal C57BL/6 blastocysts. The Cr1 produced by the normal C57BL/6 cells fully rescued the phenotype of Cr1(-/-) cells, indicating that Cr1 protein acted in a paracrine manner. Cells derived from the embryo proliferated and migrated poorly and had different adhesion properties compared to wild type. Therefore, lethality in the absence of Cr1, likely resulted largely from defective precardiac mesoderm that was unable to differentiate into functional cardiomyocytes.

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2.

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

alpha-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

Vinculin and cell-cell adhesion.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Specific arrest of cardiogenesis in cultured embryonic stem cells lacking Cripto-1.

The molecular events of cardiac lineage specification and differentiation are largely unknown. Here we describe the involvement of a growth factor with an EGF-like domain, Cripto-1 (Cr-1), in cardiac differentiation. During embryonic development, Cr-1 is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-1, we have generated Cr-1-negative embryonic stem (ES) cell lines by homologous recombination. The resulting double "knockout" ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing Cr-1 gene back into the Cr(-/-) cells. Furthermore, the lack of functional Cr-1 is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during differentiation. Differentiation into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-1 is essential for contractile cardiomyocyte formation in vitro.

Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin.

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy.

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.

Vinculin knockout results in heart and brain defects during embryonic development.

The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculin-/- embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30-40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.

Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.

UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.

Cripto: roles in mammary cell growth, survival, differentiation and transformation.

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.

Reciprocal modulation between Sp1 and Egr-1.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or CAAT box but have a region of G + C-rich sequences that appears to replace the usual promoter initiation site. The zinc-finger protein Sp1 is one of the prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar binding site and if the two sites occur in the same region, a variety of activation or inhibitory responses may be obtained. We show that competition between the two factors for overlapping sites on growth-promoting genes could explain why the overexpression of Egr-1 suppresses transformed growth in a number of cell types [Huang et al. (1995): Cancer Res 55:5054-5062; Huang et al. (1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its activity. We measured the responses of synthetic consensus binding sites and natural promoter sequences linked to a reporter gene and showed that Egr-1 inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites. In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2 cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also leads to increased Egr-1 protein expression, which further inhibits Sp1 transactivation of numerous genes. Therefore, we can account for some of the complex responses of G + C-rich enhancer/promoters by a form of "facilitated inhibition" of Sp1 by Egr-1 at overlapping sites.

Expression and function of amphiregulin during murine preimplantation development.

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-alpha significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-alpha, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-alpha in these embryos.