Androgen receptor variants with short glutamine or glycine repeats may identify unique subpopulations of men with prostate cancer.

The androgen receptor (AR) contains glutamine (CAG) and glycine (GGC) repeats that are each polymorphic in length. We screened clinically localized prostate cancers for somatic mutations in the length of the CAG and GGC repeats in the AR gene and characterized the length of these repeats in the germ-line AR gene. Somatic mutations were rare, and the range of germ-line repeat lengths in men with prostate cancer was within the range of normal in the general population. Most allele frequencies in Caucasian men with clinical prostate cancer were remarkably comparable to those in the general Caucasian population. However, a subpopulation of the men with clinical prostate cancer had a substantially higher frequency of AR alleles with 16 or 17 CAGs (6 of 59 men, 10%) than did the general population (6 of 370 alleles, 1.6%), and a different subpopulation of the men with prostate cancer had a higher frequency of AR alleles with 12 or 13 GGCs (7 of 54 men, 13%) than did the general population (1 of 110 alleles, 0.9%). Of the men with prostate cancer who had an AR gene with 16 or 17 CAGs, 83% had lymph node-positive disease, despite the lack of clinical evidence of metastatic spread. This suggests that a short AR CAG allele may be a risk factor for the development of clinically unsuspected lymph node-positive prostate cancer among men undergoing radical prostatectomy and raises the question of whether this short repeat length played an active role in the development of aggressive prostate cancer. The odds of having a germ-line AR gene with a short CAG repeat (

Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange.

The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli, and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP-containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.

Androgen-receptor gene structure and function in prostate cancer.

Androgen-receptor (AR) gene mutations have been found in clinical prostate cancer, both prior to hormonal therapy and in hormone-refractory disease that persists despite androgen-ablative therapy. Thus, mutations that are present in late-stage disease might arise prior to therapy rather than as a result of therapy. A common feature of mutations in untreated prostate cancer and in hormone-refractory prostate cancer is that the AR retains activity as a ligand-dependent transcription factor. Some AR mutations in prostate cancer show broadened ligand specificity, such that the transcription-factor activity of the AR can be stimulated not just by dihydrotestosterone (DHT) but also by estradiol and other androgen metabolites that have a low affinity for the AR. The activation of mutant AR by estrogen and weak androgens could confer on prostate cancer cells an ability to survive testicular androgen ablation by allowing activation of the AR by adrenal androgens or exogenous estrogen. Such mutations might confer an advantage even prior to androgen ablation, since prostate cancer has lower levels of 5 alpha-reductase and, therefore, of DHT, than normal. Thus, AR mutations that occur prior to therapy may characterize a more aggressive disease. A large percentage of tumors appear to have no AR gene mutation. In tumors without an AR gene mutation, AR function might be affected via other mechanisms (e.g., AR gene amplification, which could increase the amount of AR activity at a given DHT level). Importantly, the apparent absence of AR gene mutations in the majority of earlystage tumors indicates that the role of androgen in the development of clinical prostate cancer is mediated predominantly by a normal AR gene. There are actually multiple alleles of the normal AR gene; these allelic variants differ in glutamine and glycine repeat length in the transactivation domain of the protein, and they may differ in signal-transducing activity. The glutamine and glycine repeat length may thereby modulate the effect of androgen on tumor-cell proliferation that occurs during clonal expansion.

Binding sites for bacteriophage HP1 integrase on its DNA substrates.

The temperate phage HP1 integrates its genome into the chromosome of Haemophilus influenzae by site-specific recombination between host and phage DNA segments, the attachment sites. This reaction is promoted by the HP1-encoded integrase. The interactions of HP1 integrase with its DNA substrates have been characterized by DNase I footprinting. Two classes of binding sites were identified. At sites of type I, integrase binding almost completely eliminated cleavage by DNase I; type I sites shared the consensus sequence 5'-AGGGATTTWW. At type II sites, integrase binding produced alternating regions of protection from and enhancement of cleavage, suggesting that binding at these sites distorted the DNA. The consensus sequence for type II sites was 5'-ACTGGCGRTW. Each binding site contained two copies of the relevant consensus. The host attachment site (attB) contains an inverted pair of type I consensus sequences surrounding the strand exchange points. The phage attachment site (attP) includes six binding sites, three of type I and three of type II, distributed along its 500 nucleotide pairs. All type I sites contain two consensus motifs arranged as inverted repeats. One of these surrounds the strand exchange points in this substrate, one is located internally, and the third coincides with the right boundary of the attP sequence. One type II site, consisting of an inverted repeat of two type II consensus motifs, coincides with the left boundary of the attP sequence. The other two type II sites contain directly repeated pairs of the consensus and are internally located.

Microsatellite mutation (CAG24-->18) in the androgen receptor gene in human prostate cancer.

The androgen receptor (AR) gene contains a polymorphic CAG microsatellite that codes for a variable length of glutamine repeats in the AR protein. Microsatellite DNA sequences may be potential sites of genetic instability. Using the polymerase chain reaction (PCR), we screened 40 human prostate cancer specimens for expansions or deletions of this microsatellite. In one patient, nontumor DNA yielded a single PCR product, as expected for the AR, but the tumor DNA yielded two discrete products, one identical to normal, and a second smaller one. Direct sequencing revealed that the nontumor tissue contained 24 CAGs, whereas the tumor contained one fragment with 24 CAGs (wild-type) and a second fragment with 18 CAGs (mutant), representing a somatic contraction of the AR CAG repeat (CAG24-->CAG18) in the tumor. Interestingly, this patient manifested a paradoxical agonistic response to hormonal therapy with the antiandrogen flutamide.

A new structure-activity model for Ah receptor binding. Polychlorinated dibenzo-p-dioxins and dibenzofurans.

A new structure-affinity model for the aromatic hydrocarbon (Ah) receptor is reported. The proposed mathematical model completely eliminates multiple regression analysis in its formulation and overcomes the cross-class comparison inherent to classical quantitative structure-activity relationships. Taking the polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as model xenobiotics, the binding affinity of a PCDD relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is shown to be analytically related to the electron affinities, entropies, and lipophilicities of PCDD and TCDD. From the calculated dissociation constants of PCDD-Ah receptor complexes, the corresponding equilibrium constants of PCDF-Ah complexes could be computed, in agreement with the experimental observation that the trend in the binding affinities of PCDDs and PCDFs to the Ah receptor are similar. The reported model is capable of quantitatively explaining the quantitatively estimating the in vitro binding affinities of PCDDs, PCDFs, and related xenobiotics to the Ah receptor. Therefore, a halogenated aromatic compound is expected to have a higher affinity for the cytosolic protein than TCDD if it is less lipophilic and has a higher electron affinity and lower entropy. Furthermore, the affinities of structurally related polychlorinated aromatic xenobiotics for the Ah receptor could be computed from their entropies and electron affinities.