The androgenic regulation of prostate proteins with a high affinity for deoxyribonucleic acid. Evidence for a prostate deoxyribonucleic acid-unwinding protein.

1. When testosterone is injected into castrated rats in vivo, a significant increase in the incorporation of [35S]methionine into prostate proteins may be detected under conditions in vitro. 2. Studies based on DNA-cellulose chromatography show that the synthesis of prostate proteins with a high affinity for DNA is particularly enhanced by androgenic stimulation. 3. These changes in protein synthesis are negated when the anti-androgen, cyproterone acetate, is administered concomitantly with testosterone in vivo. 4. Two assays were developed for measuring the strand separation of prostate DNA; first, the retention of 3H-labelled native DNA on nitrocellulose membranes, and second, the activation of native DNA as a template for 9S prostate DNA polymerase. On the basis of these criteria, DNA-unwinding activity is present in the prostate gland and it is regulated by androgens in a steroid-and tissue-specific manner. 5. The results are discussed in the context of the mechanism of action of androgens, particularly since the changes provoked in DNA-unwinding activity by androgens precede the onset of DNA replication and mitosis.     

The control of the form and function of the ribosomes in androgen-dependent tissues by testosterone

1. The ribosome content of the rat ventral prostate gland is controlled by the concentrations of circulating androgens and the polyribosomal complement of the total population of ribosomes is acutely dependent on androgenic stimulation. After the administration of testosterone to castrated rats in vivo, there is a pronounced increase in the amounts of heavy (150-240S) polyribosomes. 2. These results are consistent with a pronounced increase in the mRNA and rRNA content of the prostate gland after the administration of testosterone in vivo. 3. From studies conducted both in vitro, the heavy prostate polyribosomes formed after androgenic stimulation are particularly active in protein synthesis. 4. The androgen-stimulated increase in the formation of prostate polyribosomes has a mandatory requirement for sustained RNA and protein synthesis. 5. Since the androgen-mediated increase in prostate polyribosomes may also be suppressed by the concomitant administration of certain anti-androgenic steroids in vivo, the response in polyribosome formation is probably initiated by the binding of a metabolite of testosterone, 5alpha-dihydrotestosterone, in the prostate gland. 6. The relevance of these findings to the pronounced increase in protein synthesis in androgen-dependent tissues after hormonal stimulation is discussed.     

A reappraisal of the effects of adenosine 3′:5′-cyclic monophosphate on the function and morphology of the rat prostate gland

1. A comparison was made of the binding of 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and cyclic AMP in the rat prostate gland. Distinct binding mechanisms exist for these compounds, and cyclic AMP cannot serve as a competitor for the 5alpha-dihydrotestosterone-binding sites and vice versa. In contrast with the results obtained with 5alpha-dihydrotestosterone, very small amounts of cyclic AMP are retained in nuclear chromatin and the overall binding of this cyclic nucleotide is not markedly affected by castration. 2. Androgenic stimulation does not lead to major increases in the adenylate cyclase activities associated with any subcellular fraction of the prostate gland. Accordingly, changes in the concentration of cyclic AMP in the prostate gland after hormonal treatment are likely to be small, but these were not measured directly. 3. When administered to whole animals in vivo, small amounts of non-degraded cyclic AMP are found in the prostate gland but sufficient to promote an activation of certain carbohydrate-metabolizing enzymes in the cell supernatant fraction. The stimulatory effects of cyclic AMP were not evident with cytoplasmic enzymes engaged in polyamine synthesis or nuclear RNA polymerases. These latter enzymes were stimulated solely by the administration of testosterone. 4. By making use of antiandrogens, a distinction can be drawn between the biochemical responses attributable to the binding of 5alpha-dihydrotestosterone but not of cyclic AMP. Evidence is presented to suggest that the stimulation of RNA polymerase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase is a consequence of the selective binding of 5alpha-dihydrotestosterone. Only the stimulation of glucose 6-phosphate dehydrogenase can be attributed to cyclic AMP or other metabolites of testosterone. 5. Overall, this study indicates that the formation of cyclic AMP is not a major feature of the androgenic response and affects only a restricted number of biochemical processes. Certainly, cyclic AMP cannot be considered as interchangeable with testosterone and its metabolites in the control of the function of the prostate gland. This difference is additionally emphasized by the failure of cyclic AMP to restore the morphology of the prostate gland in castrated animals; morphological restoration only follows the administration of androgens.     

Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg. kg(-1). day(-1)). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.     

Évolution de la cellule normale à la cellule cancéreuse prostatique hormonodépendante–hormono-indépendante

The prostatic gland is androgen-dependent. The role of androgens in the development, function and pathology of the prostatic gland (benign hypertrophy or cancer) derives from: direct evidence, resulting from experimental models (in vitro–in vivo) or from the biological analysis of normal and pathological human prostatic tissues. These data make it possible to describe the current point of our knowledge concerning the molecular, cellular, and tissular mechanisms involved; indirect data resulting from epidemiologic and clinical studies describing the impact of androgen suppression or supplementation on the prostatic gland. At the experimental level, it is generally allowed that the growth of prostate is controlled by androgens (testosterone and its metabolites). A suitable circulating testosterone level is necessary to maintain the growth, development, differentiation and function of the prostatic gland. Bilateral orchidectomy induces programmed cellular death (apoptosis) and the gland involution; exogenic testosterone administration is then able to induce the prostatic growth up to the normal level. The same applies when an impubescent animal is treated. The response of prostate to exogenic testosterone thus does not produce a growth beyond the normal volume, which is maintained by balance between proliferation and cellular death in the presence of physiological levels of androgens. The study of the mechanisms of regulation of the prostatic growth provides a fundamental justification to the chemical and hormonal treatments used by the urologists in the treatment of prostate benign hypertophy and cancer. Within the framework of the androgenic deficit related to age, a doubt persists about a potentially harmful action of the substitute androgenic treatment on prostate.     

Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.

In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.

The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme. A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors. Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.     

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

In vitro replication of bacteriophage PRD1 DNA. Metal activation of protein-primed initiation and DNA elongation.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.