Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.

Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

Transcriptional activation of the human epidermal growth factor receptor promoter by human p53.

The human epidermal growth factor receptor (EGFR) promoter is activated by both wild-type and tumor-derived mutant p53. In this communication, we demonstrate that EGFR promoter sequence requirements for transactivation by wild-type and mutant p53 are different. Transient-expression assays with EGFR promoter deletions identified a wild-type human p53 response element, 5'-AGCTAGACGTCCGGGCAGCCCCCGGCG -3', from positions --265 to --239. Electrophoretic mobility shift analysis and DNase I footprinting assays indicated that wild-type p53 binds sequence specifically to the response element. Using circularly permuted DNA fragments containing the p53-binding site, we show that wild-type p53 binding induces DNA bending at this site. We further show that the EGFR promoter is also activated by tumor-derived p53 mutants p53-143A, p53-175H, p53-248W, p53-273H, and p53-281G. However, the transactivation by mutant p53 does not require the wild-type p53-binding site. The minimal EGFR promoter from positions --104 to --20 which does not contain the wild-type p53-binding site is transactivated by the p53 mutants but not by the wild-type protein, showing a difference in the mechanism of transactivation by wild-type and mutant p53. Transactivation of the EGFR promoter by p53 may represent a novel mechanism of cell growth regulation.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Overlapping domains on the p53 protein regulate its transcriptional activation and repression functions.

Wild-type p53 has been shown to inhibit transcription from several viral and cellular promoters without known p53-binding sites, while transactivating promoters with p53-binding sites. Using a series of N- and C-terminal p53 deletion mutants and wild-type p53, we have defined the domains on p53 responsible for its transcriptional functions. To test transcriptional activation by p53 we have used a promoter-chloramphenicol acetyltransferase (CAT) construct containing synthetic p53-binding sites. To check transcriptional inhibition by p53 we have used a human cytomegalovirus immediate-early promoter construct, CMV-CAT. Using transient transfection-transcription assays in Saos-2 cells, we determined that the p53 transcriptional activation and repression domains overlap at the N-terminus. This suggests the possibility that the same transcriptional machinery is involved in both functions. A C-terminal deletion up to amino acid 327 (del 393-327) eliminated repression of CMV-CAT, while preserving the transactivation function to a large extent. Using gluteraldehyde cross-linking experiments, we observed that the mutant del 393-327, which is transactivation-competent, but repression-defective, could not oligomerize. Thus, oligomerization of p53 is not required for transactivation, but may be essential for repression. Interestingly, transactivation by the oligomerization-defective mutant could be inhibited by cotransfection with a plasmid expressing the transforming mutant p53-175H.

'Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain.

Tumor-derived p53 mutants can transcriptionally activate a number of promoters of genes involved in cellular proliferation. For this transactivation, mutant p53 does not use the wild-type p53 DNA-binding site, suggesting a mechanism of transactivation that is independent of direct DNA binding. Here we describe our analysis of the domain requirements for mutant p53 to transactivate promoters of the human epidermal growth factor receptor (EGFR), human multiple drug resistance 1 (MDR-1) and human proliferating cell nuclear antigen (PCNA) genes. We also report the identification of a structural domain required for the 'gain of function' property of mutant p53-281G. 'Gain of function' is measured as the tumorigenicity (in nude mice) of 10(3) murine cells expressing mutant p53 constitutively. We have generated internal deletion mutants of p53-281G deleting conserved domains I, II, III, IV and V, individually. We have also generated one deletion mutant eliminating amino acids 100 through 300 that removes four of the five conserved domains (II - V); another mutant, p53-281G del 393-327, deletes the oligomerization and nonsequence-specific nucleic acid-binding domains of p53. For the EGFR and MDR-1 promoters, all these mutants have significantly lower transactivation ability than intact p53-281G. These deletion mutants, however, significantly activated the pCNA promoter, suggesting that the mechanism of transactivation of the PCNA promoter is different from that of the EGFR and MDR-1 promoters. When expressed constitutively in 10(3) cells, p53-281G del 393-327 was found to be defective in inducing tumor formation in nude mice although intact p53-281G was very efficient. Thus, our results suggest that structural domains near the C-terminus are needed for 'gain of function'.

Wild-type human p53 activates the human epidermal growth factor receptor promoter.

We show that wild-type human p53 transactivates the human epidermal growth factor receptor (EGFR) promoter in vivo in a dose-dependent manner, implicating p53 in promotion of cell proliferation. This activation is sensitive to the expression of cellular oncoprotein MDM2 and human papillomavirus type 18 (HPV-18) E6 protein. The p53 response element is localized within -15 and -569 of the promoter. The EGFR promoter does not have a TATA box, and has low activity in Saos-2 cells in the absence of p53. Results from our in vivo transient transfection assays suggest that p53-binding sites, without any other known promoter element, can act as bidirectional promoters in the presence of wild-type p53. Gel retardation analyses suggest that p53 may serve to nucleate TBP on a promoter. We propose that p53 successfully nucleates the transcription complex, possibly via direct interaction with TFIID, and activates the EGFR promoter.

Genetic determinants of response to chemotherapy in transgenic mouse mammary and salivary tumors.

Several transgenic mouse tumor models were utilized to explore how specific genetic alterations affect the tumor cell response to chemotherapeutic agents in vivo. Specifically, MMTV-ras transgenic mice were interbred to p53 knock-out mice to create a model for assessing the role of p53 in chemotherapeutic responses. In addition, MMTV-ras tumors were compared to MMTV-myc and MMTV-ras/myc tumors. Mice of each genotype reproducibly develop mammary and/or salivary tumors, but tumor growth dynamics vary considerably between genotypes. MMTV-ras/p53-/- tumors exhibit higher S phase fractions than MMTV-ras/p53+/+ tumors, although both tumor types display very low apoptosis levels. In contrast, MMTV-myc tumors exhibit both high S phase fractions and spontaneous apoptosis levels. Tumor-bearing mice of each genotype were treated with either doxorubicin or paclitaxel, and effects on overall tumor growth, cell cycle distribution and apoptosis were evaluated. Surprisingly, neither agent efficiently induced apoptosis in any of the tumor models, including those with wildtype p53. Rather, tumor responses were mediated primarily by changes in cell cycle distribution. However, the spontaneous apoptosis levels did serve as a predictor of tumor growth response, in that only those tumors with high pretreatment apoptosis levels underwent significant regression following treatment with either agent.

Regulation of c-myb oncogene expression in immature and mature murine T cells.

The c-myb oncogene encodes a nuclear binding protein which may play a major role in differentiation during early T cell development. However, the functionally important transcription regions in the GC promoter site have not been defined and the significance of the regulation of this promoter site in T cell differentiation has not been determined. Therefore, the promoter strength was determined by measurement of the CAT activity in cell extracts of EL-4 cells that were transfected with a CAT expression vector that contained cloned segments of the 5' myb gene. Stepwise removal of DNA sequences between -2300 bp and -346 bp upstream from the ATG initiation codon resulted in a gradual loss of 50% of CAT activity, whereas deletion of DNA sequences from -346 to -295 and -232 to -155 bp upstream from the ATG initiation codon eliminated promoter activity. On analysis of the CAT activity after transfection of various cell lines with these same constructs, it was found that the same two promoter regions were required for high CAT activity in all the cell lines, including murine cell lines which express the alpha/beta TCR and high levels of c-myb (BW5147), the alpha/beta TCR and low levels of c-myb (Yac-1), or the gamma/delta TCR (KN 12.1 and KN 2.4 T), a murine fibroblast T cell line (NIH-3T3), and a human epithelial cell line (HeLa). However, the CAT activity did not correlate with steady state levels of expression of the c-myb gene in the murine cell lines. Our data indicate that the c-myb oncogene promoter is constitutively expressed is highly dependent on a limited region of the 5' myb gene, requires two DNA elements for optimal activity, and is functional in diverse T cell lines.

Inhibition of viral and cellular promoters by human wild-type p53.

Mutation of the p53 tumor suppressor gene is a recurring event in a variety of human cancers. Wild-type p53 may regulate cell proliferation and has recently been shown to repress transcription from several cellular promoters. We studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen promoter and on several viral promoters including the simian virus 40 early promoter-enhancer, the herpes simplex virus type 1 thymidine kinase and UL9 promoters, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus, human immunodeficiency virus type 1, and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and plasmids containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. Expression of wild-type p53 correlated with a consistent and significant (6- to 76-fold) reduction of reporter enzyme activity. A mutation at amino acid 143 of p53 releases this inhibition significantly with all the promoters studied. Expression of a p53 mutated at any one of the five amino acid positions 143, 175, 248, 273, and 281 also correlated with a much smaller (one- to sixfold) reduction of reporter enzyme activity from the herpes simplex virus type 1 thymidine kinase promoter. These mutant forms of p53 are found in various cancer cells. Thus, failure of tumor suppression correlates with loss of the promoter inhibitory effect of p53.

Activation of the human immunodeficiency virus type 1 long terminal repeat by transforming mutants of human p53.

We have studied the effects of human wild-type and mutant p53s on the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV). HeLa cells were cotransfected with a wild-type or mutant p53 expression plasmid and a plasmid containing a chloramphenicol acetyltransferase reporter gene under HIV LTR promoter control. As expected, expression of wild-type p53 inhibited promoter function. Expression of a p53 mutated at any one of the four amino acid positions 175, 248, 273, and 281 correlated with a significant increase of the HIV promoter activity. The HIV LTR was also significantly activated in Saos-2 cells that do not express endogenous p53. This finding suggests a gain-of-transactivation function by mutation of the p53 gene. Cotransfection of wild-type and mutant p53-281G expression plasmids indicated that either the wild type or the mutant was dominant in inhibiting or enhancing promoter activity, respectively, when transfected in excess of the other. Transfection experiments showed transactivation even when the Sp1, NF-kappa B, and TATA sites in the LTR were individually mutated. Synthetic minimal promoter constructs containing two Sp1 sites or two NF-kappa B sites or an ATF site are also significantly activated by the mutant p53-281G. Thus, the mutant protein may activate transcription through interaction with either a general transcription factor or a common factor that bridges the basal transcription machinery and the transcription factors Sp1, NF-kappa B, and ATF.

Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.

p53 binds to the TATA-binding protein-TATA complex.

Earlier reports show that p53, both wild type and mutants, may affect transcription. Wild-type p53 activates promoters with p53-binding sites while inhibiting promoters without binding sites. Mutant p53, on the other hand, has been shown to activate transcription from specific promoters. These observations suggest that both wild-type and mutant p53 may interact with a general transcription factor(s). In this report, we have shown that the cloned TATA-binding protein (TBP) from human and yeast interacts with human p53. TBP co-immunoprecipitates with wild-type or mutant human p53 when incubated with the p53-specific monoclonal antibody and Protein A-agarose. Wild-type murine p53 has also been found to interact with human TBP. Protein blot assays have demonstrated that the interaction between p53 and human TBP is direct. By gel retention analysis, we have shown that the complex of TBP and p53 (both wild type and mutant) can bind to the TATA box. The similar qualitative binding capability of wild-type and mutant p53 with human TBP and the similarity of the two complexes in binding to the TATA box suggest that the functional discrimination between wild-type and mutant p53 may not lie in their ability to bind TBP. The nature of the p53.TBP or p53.TBP.TATA complex may determine the success of transcription.

Analysis of the DNA-binding domain of the HSV-1 origin-binding protein.

In order to understand DNA-protein interactions at the origin of DNA replication in herpes simplex virus type 1 (HSV-1), we have undertaken an analysis of the DNA-binding domain of the origin-binding protein (OBP) and its mechanism of binding to the Oris sequence of HSV-1. Mutant DNA-binding domains were constructed, expressed in vitro, and used to test for binding by gel shift analysis. A C-terminal deletion mutant was functional in binding, thereby redefining the C-terminal boundary of the DNA-binding domain at amino acid 822. Fifteen insertion mutants were also constructed across the DNA-binding domain. Several of these mutants were unable to bind DNA. Interestingly, 4 mutants that destroy DNA binding fall within a region that has a particularly high degree of sequence similarity to the varicella zoster virus gene 51 product. A second objective was to define how the DNA-binding domain interacts with the origin. Results of gel shift analysis using contranslated proteins of different sizes suggest that the DNA-binding domain can interact with a single binding site as a monomer. Binding to the wild-type Oris template indicated that the binding domains can interact with both binding sites I and II independent of any cooperative effect mediated by the amino-termini. This suggests that the basic unit of recognition involved in OBP/Oris interactions may contain a single DNA-binding domain of OBP in association with a single binding site.

p53 and SV40 T antigen bind to the same region overlapping the conserved domain of the TATA-binding protein.

In this report we demonstrate that the cloned human TATA-binding protein (TBP) interacts with T antigen. TBP co-immunoprecipitates with T antigen when incubated with the T antigen-specific monoclonal antibody PAb419, and Protein-A agarose. Gel retention analysis with a radiolabeled TATA box-containing probe showed that the complex of TBP and T antigen can bind to the TATA box. Recently, p53 has also been shown to interact with TBP. Using TBP deletion mutants and co-immunoprecipitation experiments with p53 or T antigen, we show that both p53 and T antigen bind to the same region, amino acids 203-275, within the conserved C-terminal domain of TBP. Binding of p53 and T antigen to the same domain on TBP may lead to competition between the two proteins for transcriptional function.