Frederick B. Merk

The Structure and Function of Intercellular Junctions in Cancer

Publisher Summary Intercellular junctions are a set of structurally complex membrane components that are incorporated into the general plasma membrane at the sites of close cell-to-cell apposition. The primary function of some types of junctions, such as gap junctions, remains obscure; this is unfortunate because a considerable body of information on the occurrence, biochemical ultrastructure, and physical properties of junctions suggests that they probably do play a central role in important biological phenomena. The quantitative evaluations of the occurrence of junctions are subjective in most of the reports, although there are a few reports in which the data were obtained by quantitative electron microscopy techniques. There are a number of ways in which genes of neoplastic transformation may influence intercellular junction structure and function. Additional research is required to define the contribution of tumor gene products to the pathogenesis of junctional abnormalities in tumors and to elucidate the roles, if any, of these cell membrane defects in malignant growth.

Cytoplasmic inclusion bodies within neurons of the thalamus in myotonic dystrophy ☆: A light and electron microscope study

Abstract Abundant inclusion bodies were found within the cytoplasm of thalamic neurons in 6 patients with myotonic dystrophy, 4 of whom had mental defect. Electron microscopy studies revealed that these bodies contain a proteinacious fibrillar material surrounded by a ribosome-bearing membrane and result from coalescence of dilated cisternae of rough endoplasmic reticulum. Cytoplasmic bodies represent a structural alteration of thalamic neurons very likely related to progressive dementia, behavioral changes, hypersomnia and slow dominant posterior EEG rhythms which are manifestations of CNS dysfunction frequently encountered in patients with myotonic dystrophy.

Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization

SummaryThe sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3′ end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

Prolactin Receptor Expression in the Developing Human Prostate and in Hyperplastic, Dysplastic, and Neoplastic Lesions

In situ hybridization and immunohistochemistry were used to localize and compare the expression of the long form of the human prolactin receptor in fetal, prepubertal, and adult prostate. Results were then compared with hyperplastic, dysplastic, and neoplastic lesions. Both receptor message and protein were predominately localized in epithelial cells of the fetal, neonatal, prepubertal, and normal adult prostate. In hyperplastic lesions the expression of the receptor was unchanged with respect to normal epithelial cells. Irrespective of grade, markedly enhanced expression of the receptor was evident in dysplastic lesions. In lower Gleason grade carcinomas the intensity of receptor signal at the message and protein levels approximated that found in normal prostatic epithelium. However, in foci within higher grade cancers, receptor expression appeared diminished. Results from our study suggest that prolactin action plays a role in the development and maintenance of the human prostate and may also participate in early neoplastic transformation of the gland. Diminution of receptor expression in high grade neoplasms could reflect the emergence of a population of cells that are no longer responsive to the peptide hormone.

Role of canine basal cells in prostatic post natal development, induction of hyperplasia, sex hormone-stimulated growth; and the ductal origin of carcinoma.

The canine prostate has often been proposed as a model for abnormal growth of the human gland. Hyperplasia of the prostate is common in aging men and has been estimated to be present in 100% of old intact dogs. While prostatic carcinoma is common in older men, it appears to be rare in dogs and unlike the disease in humans, it occurs with relatively high frequency in castrated animals. Since basal cells are thought to be key participants in normal and abnormal growth of the human gland, we used immunohistochemistry to investigate the role that they may play in canine prostatic development, the evolution of hyperplasia and carcinoma, and the effects of sex hormones on these cells.

Androgen Receptor Expression in Prostatic Dysplasia (Prostatic lntraepithelial Neoplasia) in the Human Prostate: An Immunohistochemical and In Situ Hybridization Study

To evaluate the role of androgens in the pathogenesis of prostatic dysplasia, we compared the localization of androgen receptor (AR) in proliferative and nonproliferative cells in normal and dysplastic acini. Basal cells, the only proliferating cells identified in normal aani, contained AR mRNA but lacked an immunodetectable receptor. Both AR mRNA and immunodetectable receptor were present, however, in secretory and stromal cells. Androgen receptor localization in dysplastic lesions was identical to normal but here the proliferative marker Ki‐67 was found in both basal and secretory cells. Our findings suggest that androgens do not directly initiate the division of basal cells, the putative precursors of secretory cells. Instead, the hormone may act through its fully translated receptor to mainly mediate the differentiation of secretory cells. The presence of both AR and Ki‐67 in dysplastic secretory cells may indicate an abnormal direct androgen‐mediated proliferation in this compartment. This is consistent with previous evidence that secretory cell differentiation is impaired in dysplasia.

Lack of Association between Enhanced TRPM-2/Clusterin Expression and Increased Apoptotic Activity in Sex-Hormone-Induced Prostatic Dysplasia of the Noble Rat

Although the functional role of TRPM-2/clusterin in the prostate remains controversial, it has been postulated that transcriptional activation of the gene is an important mechanism in castration-induced prostatic involution and perhaps is a means for prostatic cells to escape apoptotic induction. In the present study, we have measured expression levels of TRPM-2/clusterin and apoptotic activities in the prostates of castrated Noble (NBL) rats and those treated with testosterone (T) and estradiol-17beta (E2) for 16 weeks. We have previously shown that the combined sex hormone treatment (T+E2) induces dysplasia, a purported preneoplastic lesion, exclusively in the dorsolateral prostates (DLPs) of all treated rats. In the present study, we demonstrate that, as expected, castration readily induced enhanced TRPM-2/clusterin expression, which was accompanied by increased apoptotic activity in the epithelia of DLP and ventral prostate (VP). The increase in TRPM-2/clusterin expression appeared earlier and was more dramatic in the VP than in the DLP. In sharp contrast, treatment of rats with T+E2 for 16 weeks induced augmentation of TRPM-2/clusterin expression selectively in the dysplastic lesions of the DLP but not in the lesion-free VP. The enhanced expression of TRPM-2/clusterin in the dysplastic epithelium was, however, not attended by an increase in apoptotic activity within the lesion. Thus, the observed up-regulation of TRPM-2/clusterin expression in the dysplastic foci of T+E2-treated rats occurred in animals whose androgen status remained normal and, despite the increased level of expression of this gene, apoptotic activity in these lesions was unchanged from basal values measured in the DLPs of untreated rats. These findings suggest that TRPM-2/clusterin expression in dysplastic lesions was no longer repressed by androgen nor was it associated with apoptosis. We propose that overexpression of the gene is likely a phenotype of neoplastic transformation. In addition, we speculate that TRPM-2/clusterin may serve as a survival factor, which could favor accumulation of transformed cells in dysplastic foci and thus promote the carcinogenic process.

Ultrastructure and in vitro growth characteristics of a transplantable rat pheochromocytoma

The transplantable pheochromocytoma in rats was characterized by high intracellular concentrations of primary catecholamines as indicated by the formaldehyde vapor‐induced fluorescence reaction. Ultrastructurally, two types of intracytoplasmic granules could be recognized. The first type (type 1), believed to contain norepinephrine, measured up to 0.3 μ in diameter and was composed of an electron‐dense core which was separated from the surrounding membrane by an irregular, wide electron‐lucent space. The second type of granule (type 2), believed to contain epinephrine, measured up to 0.2 in diameter and was composed of a less electron‐dense core which was separated from the surrounding membrane by a narrow regular electron‐lucent space. Both norepinephrine‐ and epinephrine‐type granules were present within the same cell in contrast to the normal rat adrenal medulla which contains these catecholamines in two distinct cell populations. When grown in vitro, the tumor cells were characterized by elongate branching processes and resembled the neuronal type cells which have been cultured from neuroblastomas and sympathetic ganglia.

Membrane transport of macromolecules: New carrier functions of proteins and poly(amino acids)

Abstract Two different carrier functions of macromolecules are described, namely the piggy-back transport of a cytotoxic agent (cadmium) complexed with ferritin and the dramatic increase in transmembrane transport of an enzyme (horseradish peroxidase) effected by the covalent attachment of a fragment of poly(L-lysine) (PLL). In the first case, the in vitro cellular toxicity of cadmium-containing ferritin is compared with that of CdCl2. The demonstration of a protein-mediated cadmium transport rests on the fact that toxicity is not suppressed when a chelating agent capable of binding more than the total cadmium content of ferritin is present in the medium together with ferritin, but is suppressed when cells are exposed to ferritin at 4°C, a condition known to suppress the cellular transport of protein. The toxicity of ferritin is due to its cellular uptake and intracellular digestions, leading to the release of free cadmium inside the cell. These data are discussed in the context of the piggy-back transport of DNA-drug complexes and of LDL-cholesterolester complexes. In the second case, the cellular uptake of horseradish peroxidase (HRP) is measured before and after conjugation with a MW 6,700 fragment of PLL. In the course of a 60 min. incubation at 37°C, monolayers of L-929 mouse fibroblasts take up 400 times more enzyme when the HRP is presented to cells as a PLL-HRP conjugate. Since conjugation causes a 60% loss of enzyme activity, it is concluded that the net transport of enzyme protein is 1000-fold greater than that of the unconjugated protein. This finding establishes that PLL can serve as a transport marker for proteins which are otherwise poorly taken up for lack of a specific receptor-mediated transport system. Electron microscopy demonstrates that the HRP-PLL conjugate is taken up by endocytosis; HRP reaction products are visible in vesicles and vacoules throughout the cytoplasm, including paranuclear areas. It is suggested that PLL could serve as a powerful transport marker for other enzymes.

Gap junctions in the myometrium of hypophysectomized estrogen-treated rats

Gap junctions, which appear in the myometrium at parturition, are believed to permit the flow of ions between smooth muscle-cell interiors thereby facilitating coordinated contractions. Gap junctions are not observed in thin-sectioned myometrium of immature hypophysectomized rats. Following estrogen administration, numerous gap junctions are present suggesting that this hormone is capable of promoting electrotonic coupling in uterine smooth muscle.