p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion.

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.

p27-p16 Chimera: a superior antiproliferative for the prevention of neointimal hyperplasia.

Cyclin-dependent kinase inhibitors (CDKi's) may be useful to treat hyperproliferative vascular disorders, such as restenosis induced following angioplasty or vein engraftment. We have shown that a novel fusion protein of the CDKi's p27 and p16, named W9, significantly reduces proliferation of human coronary smooth muscle cells in vitro, by blocking cell proliferation without inducing apoptosis. We have now evaluated the efficacy of adenovirus-mediated gene transfer of W9 (AV-W9) in a balloon-injury model, in the carotid arteries of cholesterol-fed rabbits. We observed that intravascular delivery of 2 x 10(11) viral particles of AV-W9 3 days following balloon injury inhibited intimal hyperplasia by 60% compared to a control virus (P > 0.001). PCNA expression in the AV-W9-treated vessels, a marker of injury-induced cell proliferation, was also reduced compared to the control virus-treated vessels. Direct comparison of the efficacy of AV-W9 and AV-p16 and AV-p27 in this model indicated that delivery of either of the parental genes was significantly less effective in inhibiting intimal thickening compared to the AV-W9 treatment. We conclude that combining the activities of multiple cell cycle regulatory proteins greatly increases the potency of cytostatic gene therapy in the treatment of balloon injury-induced intimal hyperplasia and represents a promising potential approach to preventing postangioplasty restenosis.

Interaction trap/two-hybrid system to identify interacting proteins.

The parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two-hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.

Interaction trap/two-hybrid system to identify interacting proteins.

This unit presents protocols designed to detect interacting proteins. Using yeast as a "test tube" and transcriptional activation of a reporter system, interacting proteins can be identified. The system can also be used to test complex formation for proteins for which there exists a reason to expect interaction.

Interaction trap/two-hybrid system to identify interacting proteins.

The parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two-hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.

Conversion of mRNA into double-stranded cDNA.

Enzymatic conversion of mRNA into double-stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide-primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt-ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The Alternate Protocol is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The Alternate Protocol takes advantage of a linker-primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt-ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5-methyl-dCTP in the first-strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI/XhoI-digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker-primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.

Interaction trap/two-hybrid system to identify interacting proteins.

The parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two-hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.

Novel chimeric p16 and p27 molecules with increased antiproliferative activity for vascular disease gene therapy.

We describe the construction and characterization of a series of novel cyclin-dependent kinase inhibitors with increased antiproliferative activity for use in the genetic treatment of hyperproliferative cell disorders, such as angioplasty-induced restenosis. These inhibitors were generated through the fusion of truncated versions of the p27 gene to the full-length p16 gene. Biochemically, the p27-p16 chimeric molecules were of comparable potency to the parental p27 in inhibiting the activities of several cyclin-dependent kinases in vitro. Replication-deficient adenoviruses encoding the parental p16, p27 genes, or their derivatives were created to assess the potency of the novel cyclin-dependent kinase inhibitor chimeric molecules to inhibit vascular smooth muscle cell proliferation, which is the seminal event in the restenosis process. One of the p27-p16 chimeric molecules, W9, was observed to be the most potent inhibitor of human primary smooth muscle and endothelial cell proliferation when compared to the p16, p27, p27 derivatives or several alternative p27-p16 chimeric molecules. Overexpression of the W9 chimeric molecule in human coronary artery smooth muscle cells induced human coronary artery smooth muscle cell growth arrest in G1 but did not induce cell apoptosis. Recombinant adenoviral vectors that express this W9 chimeric cyclin-dependent kinase inhibitor molecule constitute a novel potent antiproliferative agent for the treatment of restenosis.

The p53-independent tumoricidal activity of an adenoviral vector encoding a p27-p16 fusion tumor suppressor gene.

We describe here that DE1-adenovirus vectors (AV) expressing a p27-p16 fusion molecule, termed W9, induce tumor cell apoptosis when overexpressed in a wide range of tumor cell types. However, in primary human cells derived from a variety of normal tissues, AV-W9 induced minimal apoptosis. In tumor cells AV-W9 demonstrated 5- to 50-fold greater tumoricidal activity than either of the parental molecules p16 and p27. In these studies, AV-W9 elicited apoptosis independent of the p53 and Rb status of the tumor cells. In several murine tumor models AV-W9 demonstrated p53-independent antitumor activity. It completely prevented tumor formation in two ex vivo models, whereas the parental molecules resulted in partial protection. Furthermore, AV-W9 induced tumor regression or suppressed tumor growth when introduced intratumorally into preestablished tumors in mice. This effect may be mediated through tumor cell apoptosis or antiangiogenic activity of AV-W9. Thus, this novel chimeric molecule is more potent and capable of killing a broader spectrum of tumors than the parental p16 and p27 molecules independent of the tumor cell p53 and phenotype and represents a powerful new therapeutic agent for cancer gene therapy.

Interaction between Cdc37 and Cdk4 in human cells.

Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

Dimerization of the human papillomavirus E7 oncoprotein in vivo.

We have used a yeast two-hybrid system to show that human papillomavirus E7 proteins can form oligomeric complexes in vivo. The carboxyl-terminal cysteine-rich metal-binding domain is critical for this activity although amino-terminal sequences also contribute to oligomerization. Our experiments also reveal that E7 possesses an intrinsic transcription activation activity in yeast, which resides in the amino terminus of the protein.

APC binds to the novel protein EB1.

Mutations of the APC gene play a critical role in both sporadic and familial forms of colorectal cancer. The vast majority of these mutations result in the loss of the carboxyl terminus of the protein. To further elucidate the function of APC, we searched for cellular proteins that associate with its carboxyl terminus. One million human cDNA clones were screened with the use of the interaction trap two-hybrid system, and 67 clones were found to have a phenotype suggestive of an APC-interacting protein. Nucleotide sequence analysis revealed that 48 of these clones were derived from a single novel named EBI. The association of APC and EB1 proteins was confirmed with in vitro binding assays. mAbs against EB1 were then produced and used to demonstrate the association of APC and EB1 in vivo. The EB1 gene was predicted to encode a 268-amino acid protein without significant homology to proteins with known function. However, searches of nucleotide databases did identify evidence for at least two related human genes and a yeast homologue. This conservation suggests an essential function for EB1 that might provide clues to the mechanism through which APC suppresses colonic neoplasia.

Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor.

The thyroid hormone (T3) receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. To help elucidate the mechanisms that underlie this transcriptional regulation and other potential TR activities, we used the yeast interaction trap to isolate clones encoding proteins that specifically interact with the ligand binding domain of the rat TR beta. Several such proteins, called Trips (TR-interacting proteins), were isolated from independent selections carried out either in the presence or absence of T3. Surprisingly, all of the Trips were dependent on hormone for interaction with the TR, with some interacting only when T3 is present and others only when it is absent. Nearly all of the Trips also show similar ligand-dependent interaction with the retinoid X receptor (RXR), but none interact with the glucocorticoid receptor under any conditions. The sequences of three of the Trips predict specific functional roles: one is an apparent human homolog of a yeast transcriptional coactivator, one is a new member of a class of nonhistone chromosomal proteins, and one contains a conserved domain associated with ubiquitination of specific target proteins. Consistent with the pleiotropic effects of TR and RXR, several other Trips show significant amino acid sequence similarity with proteins involved in various regulatory pathways. The inherent transcriptional activity of the Trips was tested in yeast, and a chimeric protein consisting of a fusion of Trip4 to the bacterial LexA repressor protein is a relatively strong transcriptional activator. Similar LexA fusions to Trip9 and Trip10 had no transcriptional activity on their own but, when coexpressed with both TR and RXR, conferred T3-dependent activation to a reporter gene controlled by LexA binding sites. We suggest that this indirect T3 response provides a novel mechanism for hormonal activation of gene expression, and that studies of the Trips will provide important insights into the specific mechanisms of action of TRs and other receptors.

[Spinal metastasis from ovarian granulosa cell tumor--after 23 years]. / Granulosasejtes ováriumtumor spinális metastasisa--23 év után.

On admission, the 63-year-old patient suffered from spinal chord compression at the level of D. IV. vertebra. 23 years earlier she had undergone a total uterus exstirpation for ovarian tumor, with bilateral adnexectomy. The microcellular granular cell tumor potentially regarded as malignant, gave a spinal metastasis after 23 years of "dormancy". Half a year after the second spinal surgery, the growth again reached compression-size, requiring reoperation. On discussing the case, the authors also deal with the clinical signs of ovarian tumors as well as their prognosis and treatment modalities.

Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein.

RAG1 and RAG2 are lymphoid-specific genes that together induce V(D)J recombinase activity in a variety of nonlymphoid cell types. While no other lymphoid-specific factors are required to induce recombination, other factors with more widespread expression patterns have been implicated in the reaction. However, none of these factors have been cloned, and their relationship to the RAG proteins is unclear. Using the yeast two-hybrid assay, we have identified RCH1, a gene encoding a protein of molecular weight 58,000 that interacts specifically with RAG-1. The predicted Rch1 protein sequence is 47% identical to yeast SRP1, a protein associated with the nuclear envelope. A truncated form of Rch1, which retains the ability to interact with RAG-1, reduces V(D)J recombination activity in HeLa cells.

Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2.

We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases.

Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.

The tumour-suppressor gene p53 is inactivated in most human malignancies either by missense mutations or by binding to oncogenic proteins. In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification. MDM2 is an oncogene product that may function by binding to p53 and inhibiting its ability to activate transcription. Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain. The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery.

Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites.

We used the interaction trap to isolate a novel human protein that specifically interacts with Max. This protein, Mxi1 (for Max interactor 1), contains a bHLH-Zip motif that is similar to that found in Myc family proteins. Mxi1 interacts specifically with Max to form heterodimers that efficiently bind to the Myc-Max consensus recognition site. When bound to DNA by a LexA moiety in yeast, Mxi1 does not stimulate transcription. mxi1 mRNA is expressed in many tissues, and its expression is elevated in U-937 myeloid leukemia cells that have been stimulated to differentiate. These facts are consistent with a model in which Mxi1-Max heterodimers indirectly inhibit Myc function in two ways: first, by sequestering Max, thus preventing the formation of Myc-Max heterodimers, and second, by competing with Myc-Max heterodimers for binding to target sites.

Complex interaction of yeast nuclear proteins with the enhancer/promoter region of SV40.

Though highly complex enhancers found in animal cells have not been reported to occur in yeasts they are able to activate the transcription of adjacent genes in yeast cells. Saccharomyces cerevisiae expresses a large number of nuclear proteins that are able to recognize, and specifically bind to, the enhancer sequences of the SV40 animal tumor virus. The complexity of proteins that interact with different elements of the animal enhancers is similar in yeast and animal cell nuclear extracts. Most enhancer motifs, recognized by known trans-acting factors, are protected in footprinting experiments by yeast nuclear proteins.