Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.

Tumors arise from cells that have sustained genetic mutations resulting in deregulation of several of their normal growth-controlling mechanisms. Much of the research concerning the origins of cancer has focused on the genetic mutations within tumor cells, treating tumorigenesis as a cell-autonomous process governed by the genes carried by the tumor cells themselves. However, it is increasingly apparent that the stromal microenvironment in which the tumor cells develop profoundly influences many steps of tumor progression. In various experimental tumor models, the microenvironment affects the efficiency of tumor formation, the rate of tumor growth, the extent of invasiveness, and the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by paracrine signaling between epithelial tumor cells and neighboring stromal fibroblasts. In this review, we summarize recent advances in understanding the paracrine signaling interactions between epithelial cancer cells and associated fibroblasts and examine the effects of these bidirectional interactions on various aspects of carcinoma formation. We note, however, that paracrine signaling between other cell types within the carcinomas, such as endothelial cells and inflammatory cells, may play equally important roles in tumor formation and we will refer to these heterotypic interactions where relevant.

Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells.

A number of genetic mutations have been identified in human breast cancers, yet the specific combinations of mutations required in concert to form breast carcinoma cells remain unknown. One approach to identifying the genetic and biochemical alterations required for this process involves the transformation of primary human mammary epithelial cells (HMECs) to carcinoma cells through the introduction of specific genes. Here we show that introduction of three genes encoding the SV40 large-T antigen, the telomerase catalytic subunit, and an H-Ras oncoprotein into primary HMECs results in cells that form tumors when transplanted subcutaneously or into the mammary glands of immunocompromised mice. The tumorigenicity of these transformed cells was dependent on the level of ras oncogene expression. Interestingly, transformation of HMECs but not two other human cell types was associated with amplifications of the c-myc oncogene, which occurred during the in vitro growth of the cells. Tumors derived from the transformed HMECs were poorly differentiated carcinomas that infiltrated through adjacent tissue. When these cells were injected subcutaneously, tumors formed in only half of the injections and with an average latency of 7.5 weeks. Mixing the epithelial tumor cells with Matrigel or primary human mammary fibroblasts substantially increased the efficiency of tumor formation and decreased the latency of tumor formation, demonstrating a significant influence of the stromal microenvironment on tumorigenicity. Thus, these observations establish an experimental system for elucidating both the genetic and cell biological requirements for the development of breast cancer.

Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling.

Female reproductive hormones control mammary gland morphogenesis. In the absence of the progesterone receptor (PR) from the mammary epithelium, ductal side-branching fails to occur. We can overcome this defect by ectopic expression of the protooncogene Wnt-1. Transplantation of mammary epithelia from Wnt-4(-)/(-) mice shows that Wnt-4 has an essential role in side-branching early in pregnancy. PR and Wnt-4 mRNAs colocalize to the luminal compartment of the ductal epithelium. Progesterone induces Wnt-4 in mammary epithelial cells and is required for increased Wnt-4 expression during pregnancy. Thus, Wnt signaling is essential in mediating progesterone function during mammary gland morphogenesis.

Conservation of structural domains and biochemical activities of the MDM2 protein from Xenopus laevis.

The Mdm2 gene is the best known cellular regulator of p53 tumor suppressor activity. We report here the cloning and characterization of Xdm2, its homolog in Xenopus laevis. Human, mouse and Xenopus MDM2 proteins are more than 65% identical in several regions which are likely to be important for the biological activities of MDM2. Region I is sufficient for binding p53 and inhibiting its G1 arrest and apoptosis functions. Region II contains most of a central acidic region required for interaction with the L5 ribosomal protein and a putative C4 zinc finger. Region III is nearly identical from Xenopus to human and comprises the RING finger domain. We show that this structural conservation is associated with the conservation of three biochemical activities of MDM2; binding to the p53 and L5 proteins and specifically to RNA. Lastly, Xdm2 expression during early development is mainly restricted from the oocyte stage I/II to the blastula stage and is possibly independent of transcriptional activation by p53. These data as well as the utilization of Xenopus laevis to investigate the roles of MDM2 and p53 during early embryogenesis are discussed.

Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain.

The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. In certain cancers, MDM2 amplification is a common event and contributes to the inactivation of p53. The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide of p53 revealed that MDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix. The interface relies on the steric complementarity between the MDM2 cleft and the hydrophobic face of the p53 alpha helix and, in particular, on a triad of p53 amino acids-Phe19, Trp23, and Leu26-which insert deep into the MDM2 cleft. These same p53 residues are also involved in transactivation, supporting the hypothesis that MDM2 inactivates p53 by concealing its transactivation domain. The structure also suggests that the amphipathic alpha helix may be a common structural motif in the binding of a diverse family of transactivation factors to the TATA-binding protein-associated factors.

The MDM2 oncoprotein binds specifically to RNA through its RING finger domain.

BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.

p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches.

Insertion/deletion (IDL) mismatches in DNA are lesions consisting of extra bases on one strand. Here, the binding of p53 and its 14 kDa C-terminal domain to DNAs containing one or three 3-cytosine IDL mismatches was examined. Electron microscopy showed that both p53 forms bound predominantly as tetramers at the lesions while single-stranded binding proteins did not bind. Gel retardation assays showed that p53 formed highly stable complexes when the DNA contained the IDL mismatches, but only unstable complexes when the DNA lacked lesions (but did contain free ends). The highly stable complexes had a half-life of > 2 hr, suggesting that upon encountering lesions, p53 may recruit other proteins to the site, providing a signal for DNA damage.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Alternatively spliced forms in the carboxy-terminal domain of the p53 protein regulate its ability to promote annealing of complementary single strands of nucleic acids.

The carboxy-terminal domain of the p53 protein comprising amino acid residues 311 to 393 is able to promote the reassociation of single-stranded RNA or DNA into duplex hybrids. This domain is as efficient as the intact p53 protein in both the rate and the extent of the double-stranded product produced in this reaction. Both wild-type and mutant p53 proteins from cancerous cells carry out this reaction. The monoclonal antibody PAb421, which detects an epitope between residues 370 and 378, blocks the ability of p53 to reassociate single strands of RNA or DNA. Similarly, the alternative splice form of the murine p53 protein, which removes amino acid residues 364 to 390 and replaces them with 17 new amino acids, does not carry out the reassociation reaction with RNA or DNA. This is the first indication of functionally distinct properties of the alternative splice forms of p53. These results suggest that this splice alternative can regulate a p53-mediated reaction that may be related to the functions of this protein.

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.

Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein.

The p53 tumor suppressor gene product is a transcriptional activator that may be associated with its ability to suppress tumor cell growth. The acidic amino terminus of the p53 protein has been shown to contain this trans-activation activity as well as the domains for mdm-2 and adenovirus 5 E1B 55-kD protein binding. An extensive genetic analysis of this amino-terminal p53 domain has been undertaken using site-specific mutagenesis. The results demonstrate that the acidic residues in the amino terminus of p53 may contribute to, but are not critical for, this trans-activation activity. Rather, the hydrophobic amino acid residues Leu-22 and Trp-23 of human p53 are both required for trans-activation activity, binding to the adenovirus E1B 55-kD protein and the human mdm-2-p53 protein in vitro. In addition, hydrophobic residues Leu-14 and Phe-19 are crucial for the interactions between p53 and human mdm-2 (hdm-2). Hydrophobic residues Trp-23 and Pro-27 are also important for binding to the adenovirus 5 (Ad5) E1B 55-kD protein in vitro. These mutations have no impact on the ability of the p53 protein to bind to a p53-specific DNA element. These results suggest that 2-4 critical hydrophobic residues in the amino-terminal domain of the p53 protein interact with the transcriptional machinery of the cell resulting in transcriptional activation. These very same hydrophobic residues contact the hdm-2 and Ad5 E1B 55-kD oncogene products.

Two upstream elements activate transcription of a major histocompatibility complex class I gene in vitro.

Expression of major histocompatibility complex (MHC) class I genes exhibits unique tissue and developmental specificity. In an effort to study molecular mechanisms of MHC class I gene regulation, an in vitro transcription system has been established. In B cell nuclear extracts a template DNA containing the mouse H-2Ld promoter sequence accurately directed RNA polymerase II-dependent transcription of a G-free cassette. A conserved class I regulatory complex previously shown to moderately enhance promoter activity in vivo enhanced transcription in vitro by 2-3 fold. Much of this enhancement was accounted for by a 40 bp fragment within the complex, which was capable of activating a basal H-2Ld promoter in either orientation. Farther downstream, another element called site B was identified, which independently activated MHC class I transcription in vitro by 2-4 fold. Site B bound a specific nuclear factor(s) through an NF-1 binding site but not through a neighboring CCAAT site. The functional significance of site B in vivo was demonstrated in transfection experiments in which site B enhanced MHC class I promoter activity to a degree comparable to that seen in vitro. With the identification of the two upstream activators, MHC class I genes may serve as a model to study roles of sequence-specific DNA-binding proteins in transcription in vitro.