Evelyn R. Barrack

Immunohistochemical study of androgen receptors in metastatic prostate cancer. Comparison of receptor content and response to hormonal therapy.

A longstanding goal has been to determine whether androgen receptor (AR) levels could be used to predict the clinical response of metastatic prostate cancer to androgen withdrawal therapy. A major limitation of previous studies was the use of homogenized tissue, which yields an average AR content for all cells. By AR immunohistochemical study using an antibody specific for AR the authors assessed nuclear AR content specifically in the malignant epithelial cells of prostate needle biopsy specimens of 17 patients with Stage D prostate cancer. The authors found that prostate cancer contains AR‐positive and AR‐negative malignant cells before androgen withdrawal therapy, but the percentage of AR‐positive cells did not predict the time to tumor progression after therapy. There was no significant correlation between the percentage of AR‐positive malignant cells and the time to tumor progression. When patients were divided into two groups based on the median time to progression, the percentage of AR‐positive nuclei was not significantly different in poor responders versus good responders. When patients were divided into two groups based on the median percentage of receptor‐positive nuclei, Kaplan‐Meier estimates of the progression‐free interval revealed no significant difference between the group of patients with AR‐poor tumors and patients with AR‐rich tumors. Potential explanations for these results are discussed. The authors conclude that the percentage of AR‐positive nuclei is not a sufficient criterion to predict tumor behavior.

TGFβ in prostate cancer: A growth inhibitor that can enhance tumorigenicity

A common feature of cancer cells is the autocrine production of growth promoters and the loss of function of tumor suppressors. In our search for such features of prostate cancer, we discovered that transforming growth factor β1 (TGFβ1) levels are higher in prostate cancer than in normal prostate, and prostate cancer cells can activate endogenously‐produced latent TGFβ to a bioactive form. Because TGFβ1 is a potent growth inhibitor of epithelial cells, it seems paradoxical that malignant epithelial cells make high levels of a growth inhibitor. Even prostate cancer cells can be growth‐inhibited by TGFβ1, but only under specific conditions in vitro (plating at low cell density in serum‐free medium), and this response is readily disrupted by growth factors, serum, and extracellular matrix, to all of which the cells are exposed in vivo. This explains why prostate cancer cells are resistant to the growth‐inhibitory effect of TGFβ in vivo. In vivo, TGFβ1 actually enhances prostate tumor growth and metastasis, but not by affecting tumor cell proliferation directly. One possibility is that TGFβ affects the host to allow increased numbers of tumor cells to survive and produce progeny. In addition, since prostate cancer cells can still respond to TGFβ, e.g., by increased cell motility, even under conditions that prevent growth inhibition, the ability of TGFβ to enhance tumorigenicity in vivo might also occur via direct effects on the tumor cells themselves. I will discuss new developments in our understanding of TGFβ action, which provide a framework for elucidating the mechanism by which prostate cancer cells have devised a way to protect themselves from being growth‐inhibited by TGFβ1 in vivo. Since the cells retain the ability to be growth‐inhibited by TGFβ, indicating that the TGFβ receptors and signaling pathways for growth inhibition are intact, albeit inactive, it might be possible to reactivate this pathway to achieve a therapeutic benefit in vivo. Prostate 31:61–70, 1997.

Biological Properties of the Nuclear Matrix: Steroid Hormone Binding

Publisher Summary This chapter describes the biological properties of the nuclear matrix. The nuclear matrix may be defined as a residual nuclear scaffolding system with the dynamic properties that provides functional organization for the DNA. The nuclear matrix may provide a direct passageway from the interior of the nucleus to the pore complexes. During mitosis, the peripheral lamina components of the matrix depolymerize, and the matrix proteins at the points of attachment of the DNA loops appear to condense to form the core scaffolding of the metaphase chromosomes. Electron microscopy of this material showed a nuclear membrane surrounding fibrillar structures and ribonucleoprotein particles. The residual nuclear structures referred to above were complex structures composed of intact nuclear envelopes, residual nucleoli, and considerable amounts of ribonucleoprotein particles. The interaction of steroid hormones and their specific receptor proteins with the nucleus of target tissues is an essential step in the mechanism by which these hormones modulate nuclear events, such as gene expression.

Concepts related to salt resistant estradiol receptors in rat uterine nuclei: Nuclear matrix

When 3H-estradiol (0.1 μg) is injected into immature female rats, virtually all of the label that is recovered with uterine nuclei can be solubilized by 0.6 M KCl. Salt resistant uterine nuclear estrogen binding sites do not become labeled within one hour after the injection of 3H-estradiol, but these sites do exist and can be revealed when isolated nuclei are subjected to an in vitro estradiol exchange assay. These saturable, high affinity salt resistant sites appear to be associated with the uterine nuclear matrix, a residual structure of the nucleus.

Steroid hormone receptor localization in the nuclear matrix: Interaction with acceptor sites

The nuclear matrix is a conceptually attractive candidate for the site in the nucleus where steroid hormone-receptor complexes might interact to modulate DNA structure and function. We have demonstrated that in sex steroid target tissues a major proportion (50-100%) of the high affinity and steroid-specific receptors that become associated with the nucleus following hormonal stimulation are localized in the nuclear matrix. Direct cell-free binding assays confirm that this localization is due to the presence of specific acceptor sites in the matrix to which steroid-receptor complexes bind with high affinity and tissue specificity, and is not the result of spurious binding. The nuclear matrix appears to be a major site of hormone receptor binding in the nucleus, and this situation is consistent with the known ability of steroid hormones to stimulate gene transcription, a process which also appears to occur in association with the nuclear matrix.

Determination of Growth Fraction in Advanced Prostate Cancer by Ki-67 Immunostaining and Its Relationship to the Time to Tumor Progression After Hormonal Therapy

Reliable predictors of response for prostate cancer patients undergoing hormonal therapy are lacking. This study investigates the possibility that tumor proliferation rates might predict tumor behavior for these patients. The growth fraction of metastatic prostate cancer biopsy specimens obtained before androgen withdrawal therapy was evaluated by Ki‐67 antibody immunohistochemical study to determine whether a higher tumor growth fraction was associated with a shorter time to tumor progression after therapy. The percentage of Ki‐67‐positive malignant epithelial nuclei in the primary tumors of 17 patients ranged from 1.7% to 7.5% (median, 2.5%). When patients were divided into two response groups according to the median time to progression, poor responders (time to progression < 20 months) and good responders (≥ 20 months) had similar growth fractions (3.5 ± 0.5% versus 3.1 ± 0.6% Ki‐67‐positive nuclei, respectively). However, when patients were divided into two groups based on the median growth fraction, patients with a high growth fraction (> 2.5% Ki‐67‐positive nuclei) tended to have a shorter time to progression (median, 10 months) than patients with a low (< 2.5%) growth fraction (median time to progression, 25 months), although statistical significance was not reached. Despite this interesting trend, Ki‐67 immunostaining was not accurate to predict the time to progression in individual patients undergoing hormonal therapy. Reliable prediction of growth rates may require measurement of both cell proliferation and cell death rates.

Androgen-receptor gene structure and function in prostate cancer.

SummaryAndrogen-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 5α-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 early-stage 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.

The Relationship of Androgen Receptor Levels to Androgen Responsiveness in the Dunning R3327 Rat Prostate Tumor Sublines

The objective of this study was to determine whether androgen receptor levels in a transplantable animal model of prostatic adenocarcinoma correlated with androgen responsiveness of the tumor. This is the first comparative study of androgen receptor levels in 3 subcellular compartments (cytosol, nuclear salt-extractable and nuclear salt-resistant fractions) of 4 Dunning R3327 rat prostatic adenocarcinoma sublines that vary in their response to androgen ablation. Tumors were harvested from intact adult male rats in order to best approximate the human clinical setting in which receptor levels are quantitated prior to androgen ablative therapy. Only the nuclear salt-resistant (nuclear matrix) and total nuclear androgen receptor contents were significantly different among all tumor sublines. The properties of the tumors studied and their nuclear salt-resistant androgen receptor levels were as follows: H tumor--well-differentiated, slow growing, androgen-dependent, 63 +/- 11 fmol./mg. DNA; HI tumor--well-differentiated, slow growing, androgen-insensitive, 19 +/- 8 fmol./mg. DNA; G tumor--poorly-differentiated, fast growing, androgen-sensitive, 195 +/- 42 fmol./mg. DNA; and AT-2 tumor--anaplastic, fast growing, androgen-insensitive, no detectable receptors. There was no apparent quantitative relationship between androgen receptor content and tumor growth rates, which varied considerably irrespective of the androgen responsiveness of the tumor. However, there was a qualitative relationship between nuclear salt-resistant or total nuclear receptor content and androgen responsiveness. Higher levels of receptor (H and G tumor sublines) were associated with responsiveness to androgen ablation (cessation or slowing of growth, respectively), whereas lower levels of receptor (HI and AT-2 sublines) were associated with androgen insensitivity. These observations, based on relatively homogeneous tumors, may have important implications for human prostatic cancers which appear to be composed of heterogeneous cell populations.

The regulation of nuclear DNA template restrictions by acidic polymers

Abstract As presented in the Introduction it is now apparent that in heterokaryon cells cytoplasmic factors are capable of interacting with mammalian nuclei and inducing nucleic acid synthesis. These nucleocytoplasmic interactions are accompanied by a marked increase in nuclear volume, dispersion of chromatin and increase in nuclear DNA template function. To our knowledge the only factors which are capable of mimicking all of these cellular effects on isolated nuclei are specific large acidic polymers such as acidic proteins, acidic polysaccharides and ribonucleic acids. We have initiated a detailed study of the interactions of various types of acidic polymers on isolated nuclei and chromatin and our observations have formed the basis for this review. The regulation of DNA synthesis in mammalian cells may be a complex process involving multiple enzyme systems and initiating factors (see reviews by Simpson (1), Stein and Baserga (66) and Barlow (115)). Many of these regulatory processes require specific events at various phases of the cell cycle (65). Increasing attention has been focused on the possible role of nuclear acidic proteins as one of the important factors in regulating nucleic acid synthesis in the cell (66). The models which we have proposed in our studies may provide new insight into this problem.

A new method for labeling and autoradiographic localization of androgen receptors.

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.