WWOX, the common chromosomal fragile site, FRA16D, cancer gene.

Gross chromosomal rearrangements and aneuploidy are among the most common somatic genomic abnormalities that occur during cancer initiation and progression, in particular in human solid tumor carcinogenesis. The loss of large chromosomal regions as consequence of gross rearrangements (e.g. deletions, monosomies, unbalanced translocations and mitotic recombination) have been traditionally associated with the existence of tumor suppressor genes within the areas affected by the loss of genetic material. The long arm of chromosome 16 was identified as being frequently associated with structural abnormalities in multiple neoplasias, that led us to focus attention on the detailed genetic dissection of this region resulting in the cloning of the putative tumor suppressor gene, WWOX (WW domain containing Oxidoreductase). Interestingly, the WWOX gene resides in the very same region as that of the common chromosomal fragile site 16D (FRA16D). The WWOX gene encodes a protein that contains two WW domains, involved in protein-protein interactions, and a short chain dehydrogenase (SDR) domain, possibly involved in sex-steroid metabolism. We have identified the WWOX WW domain ligand as the PPXY motif confirming the biochemical activity of this domain. WWOX normally resides in the Golgi and we will demonstrate that Golgi localization requires an intact SDR. Inactivation of the WWOX gene during tumorigenesis can occur by homozygous deletions and possibly mutation, however, aberrantly spliced forms of WWOX mRNA have been observed even when one allele is still intact. The aberrantly spliced mRNAs have deletions of the exons that encode the SDR and these WWOX protein isoforms display abnormal intracellular localization to the nucleus possibly functioning as dominant negative inhibitors of full length WWOX. Thus, generation of aberrant transcripts of WWOX may represent a novel mechanism to functionally inactivate WWOX without genomic alteration of the remaining allele. In this article we will review the cloning and identification of WWOX as the target of FRA16D. In addition, we will discuss the possible biochemical functions of WWOX and present evidence that ectopic WWOX expression inhibits tumor growth.

AP-1 blockade inhibits the growth of normal and malignant breast cells.

We have previously demonstrated that basal AP-1 transcriptional activity is high in normal human mammary epithelial cells, intermediate in immortal breast cells, and relatively low in breast cancer cells. In this study we investigated whether differences in AP-1 transcriptional activity reflect differences in breast cells' dependence on AP-1 for proliferation. The cJun dominant negative, TAM-67, was used to determine the effect of AP-1 blockade on the growth of normal, immortal and malignant breast cells. We first showed that TAM-67 inhibits AP-1 activity in normal and malignant breast cells. We then determined whether this AP-1 inhibitor affected colony forming efficiency of the immortalized and malignant breast cells. The AP-1 inhibitor reduced colony formation of immortal breast cells by over 50% (by 58% in 184B5 cells and 62% in MCF10A cells), and reduced colony formation in the breast cancer cell line MCF7 by 43%, but did not reduce colony formation in the other breast cancer cell lines (T47D, MDA MB231 and MDA MB 435). We also determined the effect of AP-1 blockade on the growth of normal breast cells using a single cell proliferation assay. Using this assay, the growth of normal breast cells was extremely sensitive to AP-1 blockade, while immortal breast cells were moderately sensitive. We next directly tested the effect of TAM-67 expression on the growth of MCF7 breast cancer cells, using cells stably transfected with TAM-67 under the control of a doxycycline-inducible promoter. Upon induction, TAM-67 was expressed and AP-1 activity was inhibited in these cells. We then measured the growth of these cells in the presence or absence of TAM-67. The results of these studies show that the growth of MCF7 cells was suppressed by the AP-1 inhibitor, TAM-67. These results demonstrate that normal and immortalized breast cells, and some breast cancer cells (such as MCF7), require AP-1 to transduce proliferative signals, while other breast cancer cells (such as T47D, MDA MB 231 and MDA MB 435) do not. These studies suggest that the AP-1 transcription factor is a potential target for future agents for the prevention or treatment of breast cancer.

Forkhead homologue in rhabdomyosarcoma functions as a bifunctional nuclear receptor-interacting protein with both coactivator and corepressor functions.

In a search for novel transcriptional intermediary factors for the estrogen receptor (ER), we used the ligand-binding domain and hinge region of ER as bait in a yeast two-hybrid screen of a cDNA library derived from tamoxifen-resistant MCF-7 human breast tumors from an in vivo athymic nude mouse model. Here we report the isolation and characterization of the forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the hepatocyte nuclear factor 3/forkhead homeotic gene family, as a nuclear hormone receptor (NR) intermediary protein. FKHR interacts with both steroid and nonsteroid NRs, although the effect of ligand on this interaction varies by receptor type. The interaction of FKHR with ER is enhanced by estrogen, whereas its interaction with thyroid hormone receptor and retinoic acid receptor is ligand-independent. In addition, FKHR differentially regulates the transactivation mediated by different NRs. Transient transfection of FKHR into mammalian cells dramatically represses transcription mediated by the ER, glucocorticoid receptor, and progesterone receptor. In contrast, FKHR stimulates rather than represses retinoic acid receptor- and thyroid hormone receptor-mediated transactivation. Most intriguingly, overexpression of FKHR dramatically inhibits the proliferation of ER-dependent MCF-7 breast cancer cells. Therefore, FKHR represents a bifunctional NR intermediary protein that can act as either a coactivator or corepressor, depending on the receptor type.

'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'.

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.

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.

Simple procedure for creation of in-frame deletion mutations throughout an open reading frame.

A general method is presented for randomly mutagenizing open reading frames (ORF) to generate in-frame deletions and insertions. The protocol requires expression of the ORF of interest as a hybrid ORF-beta-galactosidase fusion protein. This allows colorimetric screening for beta-galactosidase activity during subsequent mutagenesis steps. Consequently, proteins with no suitable phenotypic selection or screening properties can be readily screened for mutations that disrupt and subsequently restore the reading frame of the hybrid protein. In addition, this system provides gene expression for subsequent biochemical analysis of the mutant proteins. The bovine papillomavirus type 1 (BPV-1) EI ORF has been mutagenized using this method as an example.