Activator protein 1 transcription factors are fundamental to v-rasHa-induced changes in gene expression in neoplastic keratinocytes.

The induction of mouse skin papillomas by initiation-promotion protocols is associated with aberrant expression of epithelial markers in the tumor mass. Similarly, initiation of mouse keratinocytes with a retrovirus encoding the v-rasHa gene (v-rasHa keratinocytes) causes characteristic alterations of epidermal gene expression (A. A. Dlugosz et at, Cancer Res., 54: 6413-6420, 1994). Because activator protein 1 (AP-1) proteins are likely targets of Ras activation, we have examined the role of AP-1 factors in v-rasHa keratinocytes. Introduction of v-rasHa into keratinocytes up-regulates c-Fos, deltaFos B, and Fra-1 transcripts and protein levels in nuclear extracts. The expression of Jun proteins is not significantly altered in v-rasHa keratinocytes. Transduction of cells with v-rasHa results in increased AP-1-dependent transcriptional activity, which is also simulated by transfection of keratinocytes with either c-Fos or deltaFos B but not Fra-1, suggesting that the up-regulation of c-Fos and deltaFos B contributes to this effect. To explore the role of AP-1 proteins in regulating keratinocyte markers in v-rasHa keratinocytes, we blocked the binding of AP-1 proteins to DNA by infecting keratinocytes with an adenovirus encoding a dominant-negative Fos mutant (A-FOS). A-FOS replaces endogenous Fos proteins in the formation of heterodimers with Jun family members and thus prevents the AP-1 transcription factor from binding to DNA. In v-rasHa keratinocytes, the A-FOS virus reversed the suppression of keratins 1 and 10 transcripts and protein, which is characteristically seen in tumors and v-rasHa keratinocytes. A-FOS also increased protein levels but reduced transcripts for the late marker, loricrin, a component of the cornified envelope. These findings indicate that AP-1 proteins are involved in the changes in gene expression that define the v-rasHa phenotype in mouse keratinocytes.

Identification of glucocorticoid receptor domains necessary for transcriptional activation of the mouse mammary tumor virus promoter integrated in the genome.

It has previously been determined that the mouse mammary tumor virus (MMTV) promoter when integrated in the genome assumes a defined chromatin structure which is disrupted upon addition of glucocorticoids. In contrast, a transiently introduced MMTV promoter has a random nucleoprotein structure. To reveal glucocorticoid receptor (GR) domains necessary for transcriptional activation of the MMTV promoter we compared the effects of mutations of the GR on transcriptional activation of the stably integrated versus transiently introduced MMTV promoter. For this purpose we generated a GR-negative cell line which has an MMTV promoter/reporter construct integrated in the genome and studied the transcriptional activation of this construct by different GR mutants introduced into the cells. Transcriptional activation of the integrated and transiently introduced promoter was achieved by the wild-type GR or a chimeric receptor in which the MR hormone-binding domain (HBD) replaced the GR HBD. In contrast, we found that deletion of the HBD of the GR or replacement of this region with the equivalent domain of the estrogen receptor produced receptors that were unable to activate the MMTV promoter integrated in the genome although these receptors efficiently activated the transiently introduced MMTV promoter. The HBD was not the sole determinant of MMTV transcriptional activation when integrated in the genome. Chimeric receptors which harbored the MR amino terminal domain or the wild-type MR were also unable to activate the integrated MMTV promoter. Taken together, these data indicate a rigid requirement for sequences in both the GR amino and the carboxy terminal domains for transcriptional activation of a hormone response element in the defined chromatin context of the MMTV promoter.

The biochemistry of cancer dissemination.

The progression of a tumor cell from one of benign delimited proliferation to invasive and metastatic growth is the major cause of poor clinical outcome of cancer patients. Recent research has revealed that this complex process requires many components for successful dissemination and growth of the tumor cell at secondary sites. These include angiogenesis, enhanced extracellular matrix degradation via tumor and host-secreted proteases, tumor cell migration, and modulation of tumor cell adhesion. Each individual component is multifaceted and is discussed within this review with respect to historical and recent findings. The identification of components and their interrelationship have yielded new therapeutic targets leading to the development of agents that may prove effective in the treatment of cancer and its metastatic progression.

Signal transduction convergence: phorbol esters and insulin inhibit phosphoenolpyruvate carboxykinase gene transcription through the same 10-base-pair sequence.

Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and cAMP increase PEPCK gene transcription and gluconeogenesis, whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant, since they prevent cAMP and glucocorticoid-stimulated transcription. Basal promoter elements and hormone response elements for cAMP, glucocorticoids, and insulin have been defined in previous studies. By using stable transfectants containing a variety of different PEPCK-chloramphenicol acetyltransferase fusion gene constructs, a phorbol ester response sequence, located between positions -437 and -402 relative to the transcription start site, was identified. This region coincides with the insulin response sequence that has recently been defined in the PEPCK promoter. Using a vector containing various wild-type and mutated sequences of this region ligated to the heterologous thymidine kinase promoter, we delineated the boundaries of both elements to the 10 base pairs between positions -416 through -407. Thus, although it has been previously shown that insulin and phorbol esters repress PEPCK gene transcription through distinct pathways, the final target of insulin and phorbol ester action is the same DNA element.

Dual start motif in two lambdoid S genes unrelated to lambda S.

The lysis gene region of phage 21 contains three overlapping reading frames, designated S21, R21, and Rz21 on the basis of the analogy with the SRRz gene cluster of phage lambda. The 71-codon S21 gene complements lambda Sam7 for lysis function but shows no detectable homology with S lambda in the amino acid or nucleotide sequence. A highly related DNA sequence from the bacteriophage PA-2 was found by computer search of the GenBank data base. Correction of this sequence by insertion of a single base revealed another 71-codon reading frame, which is accordingly designated the SPA-2 gene and is 85% identical to S21. There are thus two unrelated classes of S genes; class I, consisting of the homologous 107-codon S lambda and 108-codon P22 gene 13, and class II, consisting of the 71-codon S21 and SPA-2 genes. The codon sequence Met-Lys-(X)-Met...begins all four genes. The two Met codons in S lambda and 13 have been shown to serve as translational starts for distinct polypeptide products which have opposing functions: the shorter polypeptide serves as the lethal lysis effector, whereas the longer polypeptide acts as a lysis inhibitor. To test whether this same system exists in the class II S genes, the Met-I and Met-4 codons of S21 were altered in inducible plasmid clones and the resultant lysis profiles were monitored. Elimination of the Met-1 start results in increased toxicity, and lysis, although not complete, begins earlier, which suggests that both starts are used in the scheduling of lysis by S21 and is consistent with the idea that the 71- and 68-residue products act as a lysis inhibitor and a lysis effector, respectively. In addition, the R gene of 21 was shown to be related to P22 gene 19, which encodes a true lysozyme activity, and was also found to be nearly identical to PA-2 ORF2. We infer that the 21 and PA-2 R genes both encode lysozymes in the T4 e gene family. These three genes form a second class lambdoid R genes, with the lambda R gene being the sole member of the first class. The existence of two interchangeable but unrelated classes of S genes and R genes is discussed in terms of a model of bacteriophage evolution in which the individual gene is the unit of evolution.