p53 mutations in prostate cancer bone metastases suggest that selected p53 mutants in the primary site define foci with metastatic potential.

PURPOSE: This study was undertaken to establish the pattern of specific p53 gene mutations in prostate cancer within primary tumors and distant metastases. MATERIALS AND METHODS: We performed polymerase chain reaction-single-strand conformation polymorphism and sequencing analyses of p53 exons 5-8 in DNA extracted from 22 formalin-fixed, paraffin-embedded tissues from 17 patients. Samples from three patients included specimens from primary and metastatic sites (paired specimens). RESULTS: G:C-to-A:T transitions were the most common point mutations (64%). Six (55%) of 11 G:C-to-A:T transitions occurred at CpG dinucleotides in five hot-spot codons (175, 245, 248, 273, and 282). Sequencing analysis of the paired samples revealed that two of the three pairs had the same mutation in both. Sequencing analysis of DNA from a different area of one of the primary tumors revealed a different mutation in the p53 gene. CONCLUSIONS: Our results suggest that specific p53 mutations participate in the progression of human prostate cancer. These findings support those of others that indicate that the primary cancer is heterogeneous and clonal expansion occurs during the progression of clinically detectable prostate cancer. Our data also imply that p53 mutations at the primary site may be predictive of metastases.

Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene.

Malignant mesothelioma is one of the very few extrarenal neoplasms in which the Wilms tumor suppressor gene (wt1) is expressed. We examined wt1 for alterations in rat mesotheliomas, a well characterized animal model for the human disease. Southern analysis revealed a 3.5 kb EcoRI wt1 fragment readily detectable in majority of mesothelioma cell lines and primary mesotheliomas but not in normal rat tissues. Cloning and sequencing of this fragment revealed that the presence of this EcoRI fragment resulted from an inability of this enzyme to cut at a EcoRI site in intron 1 of wt1. This site contains potential motifs for cytosine methylation and treatment of mesothelioma cells with 5-azadeoxycytosine restored the normal EcoRI digestion pattern of wt1 in these cells indicating that cleavage was inhibited by methylation at this site. Southern analysis using HpaII/MspI digestion revealed no differences in methylation between mesothelioma cell lines and normal mesothelium at other CpG sites in wt1 5' region. Renal cell carcinoma lines which did not express wt1 were also methylated at this EcoRI site. Our identification of a site frequently methylated in malignant cells, independent of gene expression, provides a new model system to study determinants of site-specific methylation in tumors.

Selective conservation of GAP-43 structure in vertebrate evolution.

GAP-43 (a.k.a. B-50, F1, pp46, or neuromodulin) is a major growth cone membrane protein whose expression is widely correlated with successful axon elongation, but whose function remains unknown. To distinguish the structural features of GAP-43 most relevant to its cellular functions, we have determined features of the protein that are most highly conserved in vertebrate evolution. Comparison of fish and mammalian GAP-43 distinguishes two domains of the protein. A strictly conserved amino-terminal domain contains the putative site for fatty acylation and membrane attachment, a calmodulin binding domain, and a proposed phosphorylation site. In the much larger carboxy-terminal domain, amino acid composition is strongly conserved without extensive sequence conservation. This amino acid composition predicts an extended, negatively charged rod conformation with some similarity to the side arms of neurofilaments. The results suggest that the biological roles of GAP-43 may depend on an ability to form a dynamic membrane-cytoskeleton-calmodulin complex.

Ribosome-associated estradiol-binding components in the uterus and their relationship to the translational capacity of uterine ribosomes.

Estradiol transiently increases the rate of peptide elongation on uterine ribosomes from ovariectomized mature rats during the first 2 h after hormone injection, suggesting the existence of direct or indirect estradiol receptor interaction with ribosomes. Characterization of estradiol-binding components on isolated uterine ribosomes, microsomes, and cytosol under identical assay conditions indicated that microsomes and cytosol contain estradiol-binding components with similar affinities for estradiol (Kd = 0.5 nM) and sucrose gradient sedimentation characteristics (3.8S and 5.2S for preparations incubated at 0 and 30 C for 1 h, respectively). Those on ribosomes exhibited a higher affinity for estradiol (Kd = 0.14 nM) and had heterogeneous and more dense sedimentation characteristics (5.5-6.0S). The ribosome-associated estradiol binder was clearly different from transformed cytosol and nuclear estradiol receptors based on sedimentation characteristics under identical conditions. Like cytosol and nuclear receptors, microsomal and ribosomal estradiol binding underwent exchange reactions in vitro at 30 C, but not at 0 C. All in vitro bound, but not all in vivo bound, [3H] estradiol could be exchanged from microsomes or ribosomes by estradiol. [3H]Estradiol could be exchanged from ribosomes by a variety of estrogens, but not by progestins, glucocorticoids, or androgens. The amount of estradiol-binding activity on ribosomes decreased after estradiol administration in vivo and was inversely correlated with the rate of peptide elongation by the ribosomes in a cell-free protein synthesis system. These results suggest that accumulation of an estradiol-binding protein, perhaps a nascent estradiol receptor, on ribosomes in the absence of in vivo estradiol may directly or indirectly inhibit the peptide elongation reaction.

Effect of 3-hydroxy-3-methylglutaric acid on cholesterol metabolism in female Japanese quail.

The compounds 3-hydroxy-3-methylglutaric acid (HMG) and HMG diethylester were administered to laying Coturnix hens to evaluate their potential in reducing yolk cholesterol concentrations. The administration of HMG did not produce consistent differences from controls in tissue levels of either cholesterol or triglycerides. The administration of HMG diethylester appeared to increase the de novo synthesis of cholesterol and the mobilization of cholesterol from liver to serum, as assayed by the uptake and conversion of [1-14C]acetate into 14C-cholesterol. Neither HMG diethylester nor HMG significantly decreased the amount of cholesterol deposited in egg yolk.

Effects of diazacholesterol, triparanol, and beta-sitosterol on egg cholesterol deposition in coturnix quail.

Studies were undertaken to determine the effect of inhibitors of cholesterol synthesis on deposition of cholesterol in eggs of Japanese quail. Results indicate that this bird responds similarly to the laying hen, making it a useful screening device for these types of compounds. Administration of either triparanol or 20,25 diazacholesterol resulted in a decreased cholesterol content of the yolk. Concomitant with this decrease was an increase in desmosterol deposition. Beta sitosterol (2%) fed either alone or with lecithin (2%) did not result in a decrease in egg yolk cholesterol. No beta-sitosterol was found in the egg yolk. Diazacholesterol fed either with sitosterol, or sitosterol plus lecithin, was not effective in reducing the total sterol content of egg.