Donald S. Coffey

The development of human benign prostatic hyperplasia with age

In this study we report the prevalence and growth rate of human benign prostatic hyperplasia with age by combining and analyzing data from 10 independent studies containing more than 1,000 prostates. The normal prostate reaches 20 plus or minus 6 gm. in men between 21 and 30 years old, and this weight remains essentially constant with increasing age unless benign prostatic hyperplasia develops. The prevalence of pathological benign prostatic hyperplasia is only 8 per cent at the fourth decade; however, 50 per cent of the male population has pathological benign prostatic hyperplasia when they are 51 to 60 years old. The average weight of a prostate that is recognized at autopsy to contain benign prostatic hyperplasia is 33 plus or minus 16 gm. Only 4 per cent of the prostates in men more than 70 years old reach sizes greater than 100 gm. An analysis of a logistic growth curve of benign prostatic hyperplasia lesions removed at prostatectomy indicates that the growth of benign prostatic hyperplasia is initiated probably before the patient is 30 years old. The early phase of benign prostatic hyperplasia growth (men between 31 and 50 years old) is characterized by a doubling time for the tumor weight of 4.5 years. In the mid phase of benign prostatic hyperplasia growth (men between 51 and 70 years old) the doubling time is 10 years, and increases to more than 100 years in patients beyond 70 years old.

IDENTIFICATION OF A NUCLEAR PROTEIN MATRIX

Abstract The structural framework of the rat liver nucleus has been identified and consists of a nuclear protein matrix. This matrix is 98.4% protein, 0.1% DNA, 1.2% RNA, and 0.5% phospholipid. The nuclear protein matrix is composed primarily of three acidic polypeptide fractions in the molecular weight range of 60–70,000 daltons.

A fixed site of DNA replication in eucaryotic cells

Abstract We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with 3 H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.

Supercoiled loops and eucaryotic DNA replication

Abstract Recent studies have indicated that the DNA of the eucaryotic nucleus is organized in the form of supercoiled loops. We show here that after depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact (unnicked) loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix. This halo of DNA loops can be reversibly wound using various concentrations of ethidium bromide and is irreversibly unwound when the DNA is gently nicked with DNAase I or exposure to ultraviolet light. These structures, comprising a halo of unwound DNA loops anchored to a nuclear matrix, provided us with a way in which to examine the relationship of DNA replication to the DNA loops. 3T3 cells were pulse-labeled with 3 H-thymidine for various periods and autoradiography was performed on the matrix-halo structures. It was found that after a 1 min pulse, >80% of the autoradiographic grains representing newly replicated DNA were found within the nuclear matrix even though the surrounding halo region contained 80% of the total nuclear DNA. With longer pulse times, the autoradiographic grains could be seen to move progressively outward from the nuclear matrix into the halo region. Over 70% of the autoradiographic grains could be chased out of the central matrix into the surrounding halo if the 1 min pulse was followed by a 1 hr incubation with excess unlabeled thymidine. These findings indicate that DNA replication occurs at fixed sites at the base of the loops and suggests that the replicating DNA loops are motile with respect to their nuclear matrix anchorage sites.

Comparison of spontaneous and experimentally induced canine prostatic hyperplasia.

Spontaneous prostatic hyperplasia in the beagle appears to progress with age from a glandular to a cystic histological appearance. Prostatic hyperplasia can be induced in young beagles with intact testes by treatment for 4 mo with either dihydrotestosterone or 5 alpha-androstane-3 alpha, 17 beta-diol, alone, or with either of these steroids in combination with 17 beta-estradiol. In contrast, the induction of prostatic hyperplasia in young castrated beagles, in which the gland had been allowed to involute for 1 mo, requires the administration of both 17 beta-estradiol and either 5 alpha-androstane-3 alpha, 17 beta-diol or dihydrotestosterone. Testosterone and 17 beta-estradiol, either singly or in combination, did not produce the hyperplastic condition in intact or castrated beagles. The experimentally induced prostatic hyperplasia is identical in pathology to the glandular hyperplasia that occurs naturally in the aging dog with intact testes. However, cystic hyperplasia was not produced by any of the treatments tested in young animals.

Considerations in the isolation of rat liver nuclear matrix, nuclear envelope, and pore complex lamina☆

Abstract A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.

Stem cell features of benign and malignant prostate epithelial cells.

PURPOSE We present a new hypothesis suggesting that the different malignant potential of benign prostatic hyperplasia (BPH) and high grade prostatic intraepithelial neoplasia may be explained by distinct alterations in stem cell-like properties. MATERIALS AND METHODS We used our results and the recent literature to develop this hypothesis in the context of an updated prostate stem cell model. RESULTS While high grade prostatic intraepithelial neoplasia is a likely precursor lesion to many prostatic adenocarcinomas, BPH rarely if ever progresses directly to carcinoma. Prostate epithelium contains basal and secretory compartments. Secretory cells appear to differentiate from basal cells. Thus, prostatic stem cells most likely reside in the basal compartment. In BPH there is a slight increase in epithelial proliferation, yet most replicating epithelial cells within BPH maintain their normal restriction to the basal compartment. In high grade prostatic intraepithelial neoplasia there is a marked increase in cell proliferation. In contrast to BPH, the majority of proliferating cells in high grade prostatic intraepithelial neoplasia reside in the secretory compartment. The biological significance of this topographic infidelity of proliferation in high grade prostatic intraepithelial neoplasia remains unclear but may relate mechanistically to down regulation of the cyclin dependent kinase inhibitor, p27kip1. Normal basal cells express GSTP1, an enzyme that inactivates reactive electrophiles and organic hydroperoxides, and that may protect cells from deoxyribonucleic acid damaging agents. In contrast, normal secretory cells and high grade prostatic intraepithelial neoplasia cells do not express this enzyme. CONCLUSIONS We propose that topographic infidelity of proliferation produces a population of secretory cells replicating in the absence of key genome protective mechanisms, thus setting the stage for an accumulation of genomic alterations and instability in high grade prostatic intraepithelial neoplasia. This action occurs along with activation of telomerase, resulting in an immortal clone capable of developing into invasive carcinoma. The model predicts that genome protection remains intact in BPH, minimizing its malignant potential.

Prostate Stem Cell Compartments : Expression of the Cell Cycle Inhibitor p27Kip1 in Normal, Hyperplastic, and Neoplastic Cells

The stem cells of rapidly renewing tissues give rise to transiently proliferating cells, which in turn give rise to postmitotic terminally differentiated cells. Although the existence of a transiently proliferating compartment has been proposed for the prostate, little molecular anatomical evidence for its presence has been obtained to date. We used down-regulation of the cyclin-dependent kinase inhibitor p27Kip1 to identify cells capable of entering the proliferative phase of the cell cycle and, therefore, competent to fulfill the role of the transiently proliferating compartment. We examined the expression of p27Kip1 in relation to its role in the development of prostatic carcinoma. Formalin-fixed paraffin-embedded specimens from matched samples of normal-appearing prostate tissue, benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, primary adenocarcinomas, and pelvic lymph node metastases were evaluated by comparative immunohistochemistry against p27Kip1. In normal-appearing prostate epithelium, moderate to strong nuclear staining of p27Kip1 was present in greater than 85% of the terminally differentiated secretory cells. The normal basal cell compartment, believed to contain prostatic stem cells, showed distinctive p27Kip1 expression; acini in epithelial benign prostatic hyperplasia tissue contained more p27Kip1-negative basal cells than acini from non-benign prostatic hyperplasia tissue. A third layer of cells was identified that was sandwiched between the basal cells and the luminal cells, and this layer was consistently p27Kip1 negative. This intermediate layer was accentuated in the periurethral region, as well as in prostate tissue that had been subjected to prior combined androgen blockade. We hypothesize that, on appropriate additional mitogenic stimulation, cells in this layer, and other p27Kip1-negative basal cells, are competent for rapid entry into the cell cycle. Consistent with the fact that cancer cells are capable of cell division, all cases of high-grade prostatic intraepithelial neoplasia and invasive carcinoma also showed down-regulation of p27Kip1 as compared with the surrounding normal-appearing secretory cells. In pelvic lymph node metastases, p27Kip1 expression was also reduced. In summary, our results suggest that lack of nuclear p27Kip1 protein may delineate a potential transiently proliferating subcompartment within the basal cell compartment of the human prostate. In addition, these studies support the hypothesis that reduced expression of p27Kip1 removes a block to the cell cycle in human prostate epithelial cells and that dysregulation of p27Kip1 protein levels may be a critical early event in the development of prostatic neoplasia.

A Structural Analysis of the Role of the Nuclear Matrix and DNA Loops in the Organization of the Nucleus and Chromosome

SUMMARY The interphase nucleus is characterized by a nuclear matrix structure that forms a residual scaffolding composed of approximately 10% of the total nuclear proteins. The nuclear matrix contains residual elements of the pore-complex and lamina, the nucleolus, and an intranuclear fibrous network that provides the basic shape and structure of the nucleus. In the interphase nucleus this nuclear matrix has been reported to be a central element in the organization of DNA loop domains and to contain fixed sites for DNA replication and transcription. In this study, we have analysed the role of the nuclear matrix and the DNA loop domains in the organization and structure of the number 4 human chromosome. A model is proposed that closely approximates the observed structural dimensions of this chromosome. The model is composed of 30 nm diameter filaments formed from a solenoid of six nucleosomes per turn. This 30 nm solenoid filament is organized as loops of DNA each containing approximately 60 000 base-pairs; each loop is anchored at its base to the nuclear matrix. A radial loop model containing 18 of these loops per turn forms a new unit of chromosome structure termed the miniband. Approximately 106 of these minibands are arranged along a central axis to form the final chromatid. The role of the nuclear matrix in this organization is presented. The accuracy of the proposed model is tested by comparing its features with the known properties of the number 4 human chromosome.

Similarities of prostate and breast cancer: Evolution, diet, and estrogens

Environment determines the risk of both prostate and breast cancer, and this risk can vary >10-fold. In contrast, no risk exists for human seminal vesicle cancer demonstrating tissue specificity. There is also species specificity, because there is no risk for prostate cancer in any other aging mammal except the dog. A study of evolution indicates that the prostate and breast appeared at the same time 65 million years ago with the development of mammals. All male mammals have a prostate; however, the seminal vesicles are variable and are determined by the diet so that species primarily eating meat do not have seminal vesicles. The exception is the human, who has seminal vesicles and consumes meat, although this is a recent dietary change. Human lineage departed from other higher primates 8 million years ago. The closest existing primate to humans is the bonobo (pigmy chimpanzee), which does not eat meat but exists primarily on a high fruit and fresh vegetable diet. Homo sapiens evolved only about 150,000 years ago, and only in the last 10% of that time (10 to 15 thousand years ago) did humans and dogs dramatically alter their diets. This is the time when humans domesticated the dog, bred animals, grew crops, and cooked, processed, and stored meats and vegetables. All current epidemiologic evidence and suggestions for preventing prostate and breast cancer in humans indicates that we should return to the original diets under which our ancestors evolved. The recent development of the Western-type diet is associated with breast and prostate cancer throughout the world. It is believed that the exposure to and metabolism of estrogens, and the dietary intake of phytoestrogens, combined with fat intake, obesity, and burned food processing may all be related to hormonal carcinogenesis and oxidative DNA damage. An explanatory model is proposed.