Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C.

Germ-line mutations of the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of human tumors, and somatic mutations of this gene have been identified in sporadic renal cell carcinomas and cerebellar hemangioblastomas. Two transcriptional elongation factors, Elongin B and C, were shown to bind in vitro and in vivo to a short, colinear region of the VHL protein (pVHL) that is frequently mutated in human tumors. A peptide replica of this region inhibited binding of pVHL to Elongin B and C whereas a point-mutant derivative, corresponding to a naturally occurring VHL missense mutation, had no effect.

Tumour suppression by the human von Hippel-Lindau gene product.

A partial cDNA sequence for the gene linked to the von Hippel-Lindau (VHL) syndrome was reported in 1993. Mutation or loss of both VHL alleles has been documented in sporadic renal cell carcinomas and in the neoplasms that arise in von Hippel-Lindau kindreds. We have determined that the protein product of the VHL gene is an approximately 30 kilodalton cytoplasmic protein.

Transcriptional control by E2F.

Considerable evidence suggests that the E2F/DRTF1 family of transcription factors (hereafter referred to as 'E2F') plays a critical role in cell growth control. For example, the ability of several small DNA tumour viruses, such as SV40, adenovirus and human papillomavirus, to transform certain cells is tightly linked to their ability to deregulate E2F. Furthermore, E2F appears to directly regulate the transcription of a diverse set of genes implicated in DNA replication and cell growth control.

The transcription factor E2F-1 is a downstream target of RB action.

Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions.

Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis.

E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53.

Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter.

The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance.

Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase.

Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting transcription factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.

Cloning, chromosomal location, and characterization of mouse E2F1.

E2F has been implicated in growth control because of its association with the retinoblastoma protein and the presence of E2F binding sites in the promoters of several growth-regulated genes. Proteins that bind to an E2F site have been cloned from human and mouse cells. However, these two proteins (human E2F1 and mouse DP-1) are quite different in sequence.

Structural and functional contributions to the G1 blocking action of the retinoblastoma protein. (the 1992 Gordon Hamilton Fairley Memorial Lecture).

The retinoblastoma gene product (RB) contributes to normal cell growth control. One of its functions is manifest as a block to exit from G1, which is carried out by an RB subspecies which is un- or underphosphorylated. After RB phosphorylation, a process which occurs towards the end of G1 in cycling cells, the block is lifted allowing a cell to enter S.

E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product.

Previous studies have shown that the carboxyl-terminal region of E2F-1 (residues 368-437) can support transcriptional activation when linked to the DNA-binding domain of the yeast transcription factor GAL4. This region also contains an 18-residue retinoblastoma (RB)-binding sequence, raising the possibility that RB binding might inhibit the ability of E2F-1 to form protein-protein contacts required for activation. Here we report a further analysis of the E2F-1 activation domain.

Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein.

The retinoblastoma gene product (Rb) is a nuclear phosphoprotein that regulates cell cycle progression. Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation. In this report, it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation.

Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties.

An expression vector was modified to permit the rapid synthesis of purified, 32P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen lambda gt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB "pocket" is known to bind, directly or indirectly, to the cellular transcription factor, E2F, implicated in cell growth control.

Identification of a growth suppression domain within the retinoblastoma gene product.

To date, all naturally occurring retinoblastoma susceptibility gene (RB) mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the "pocket" domain). This domain extends from residue 379 to 792. When full-length RB and certain truncated forms were synthesized in human RB -/- cells, we found that the minimal region necessary for overt growth suppression extended from residue 379 to 928.

Pyomyositis in patients with diabetes mellitus.

Pyomyositis is a pyogenic infection of skeletal muscle that is endemic in the tropics and is being recognized with increasing frequency in temperate climates. We report two cases of nonendemic pyomyositis in patients with diabetes mellitus. A review of the literature suggests that diabetes mellitus may be an important risk factor for the development of pyomyositis.

The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein.

A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNA-binding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNA-binding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the transcription factor E2F or with a protein having closely related DNA-binding properties.

Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product.

The SV40 T antigen (T)/adenovirus E1A-binding domain of the retinoblastoma gene product (pRB) has been fused to S. japonicum glutathione S-transferase, and the chimera, bound to insoluble glutathione, was used to search for cellular proteins that can interact specifically with pRB. At least seven such proteins were detected in extracts of multiple human tumor cell lines.

Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product.

It has previously been demonstrated that the simian virus 40 large T antigen and adenovirus E1A proteins can form complexes with the retinoblastoma susceptibility gene product (RB). We studied the ability of these proteins to bind to mutant RB proteins in vitro. A region of RB spanning residues 379 to 792 was found to be both necessary and sufficient for binding to T or E1A.

Systemic lupus erythematosus and myelofibrosis.

Acute thrombocytopenia developed in a 27-year-old woman with systemic lupus erythematosus (SLE). Antibodies to red cells and granulocytes were identified in addition to an increase in platelet-associated IgG. Serum complement levels were reduced, circulating immune complexes were present, and high titers of antinuclear antibodies and antibodies to native DNA were demonstrated.

Lymphoscintigraphy.

Lymphoscintigraphy has been used as a noninvasive means of evaluating lymph node involvement. The potential exists for its wider application to other malignancies. Additionally, it is useful in determining the etiology of edema of the extremities.

Blood flow to primary tumors and lymph node metastases in SMT-2A tumor-bearing rats following intravenous flunarizine.

Tumor blood flow is an important determinant of the efficacy of presently available antineoplastic treatment modalities. Using 113Sn-labeled microspheres, 25 micron in diameter, we measured blood flow to primary tumors and regional lymph node metastases in conscious SMT-2A mammary adenocarcinoma-bearing syngeneic rats following a single i.v.

Microwave hyperthermia and its effect on tumor blood flow in rats.

The present experiments were designed to investigate the effects of microwave hyperthermia on the microcirculation of normal and malignant tissues in female Wistar/Furth rats. During the localized heating of anesthetized rats, the hind leg musculature surrounding the SMT-2A mammary adenocarcinoma was heated at either 39 degrees, 42 degrees, or 44 degrees. Neither the body temperature, cardiac output, heart rate, nor systemic arterial pressure were significantly altered by heating at these temperatures for up to 60 min.

Effect of verapamil on malignant tissue blood flow in SMT-2A tumor-bearing rats.

We investigated the influence of the calcium antagonist verapamil on malignant and normal tissue blood flow using 25-micrometer 113Sn-labeled microspheres. Isogeneic Wistar-Furth rats were inoculated with a metastasizing mammary gland adenocarcinoma (SMT-2A) in the hindlimb musculature and mammary gland. Verapamil was administered as an i.