Current Topics in Experimental Endocrinology: Volume 3

Current Topics in Experimental Endocrinology, Volume 3 covers the relationship between the endocrine system and some types of tumors. The book discusses the perspectives, pitfalls, and potentials of tissue culture in endocrine research; the tumor types associated with ectopic adrenocorticotropin hormone secretion, particularly nonendocrine tumors; and the hormonal control of breast cancer growth in women and rats. The text also describes the status of steroid receptors in breast tumors; the physiopathological aspects of prolactin secretion in patients with pituitary tumors; and the biochemical endocrinology of prostatic tumors. The ectopic production of human chorionic gonadotropin and its alpha- and beta-subunits is also considered. Endocrinologists, oncologists, chemists, gynecologists, and students taking related courses will find the book invaluable.

• Language: English
• Publisher: Academic Press
• Release date: Oct 22, 2013
• ISBN: 9781483217352

Book preview

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Preface

L. Martini and V.H.T. James

This is the third volume of Current Topics in Experimental Endocrinology, a series initiated in 1971. The two previous volumes were collections of unrelated chapters covering different aspects of modern endocrinology. In the preparation of this volume, a different approach has been followed. After extensive consultations, the Editorial Board and the two Editors have decided to center the whole book on a single and controversial topic: the relationship between the endocrine system and some types of tumors. It is felt that this approach will provide the reader with an up-to-date synthesis of an area of research that is rapidly expanding and that may have important therapeutic and diagnostic implications in the future.

Included are chapters on basic research in this area as well as more clinically oriented chapters. All chapters have been prepared by internationally recognized authorities in their respective fields, who have covered the topics assigned to them in an exhaustive and critical fashion.

We hope that this new policy of the Editorial Board will meet with the approval of the readers. We wish to extend our warmest thanks to the participating scientists who have accepted the time-consuming task of summarizing their own results and reviewing in detail the impressive literature already existing in this field.

Tissue Culture in Endocrine Research: Perspectives, Pitfalls, and Potentials

Michael J. O’Hare, Morag L. Ellison and A. Munro Neville,     Unit of Human Cancer Biology, Ludwig Institute for Cancer Research, In Conjunction With Royal Marsden Hospital, Sutton, Surrey, England

Publisher Summary

This chapter discusses tissue culture’s role in some selected chemical, pathological, and fundamental aspects of the endocrinology of both steroid and peptide hormone-secreting tissues. Tissue culture has many forms; none is easy and many are both time-consuming and laborious. In some respects, they still represent art forms with a complexity that defies simple analysis. The chapter describes some of the reasons why this has occurred. If, however, tissue culture is to achieve preeminence in experimental studies, its days as an art rather than a science must be numbered. Certain basic problems of methodology, however, still remain to be solved by systematic experimentation, leading ultimately to the preservation of complete structural and functional integrity of purified endocrine cells in culture under completely defined conditions. Further progress in the use of tissue culture to study functional activity and growth may be considerably hampered until these requirements are fulfilled. It would be foolhardy to suppose, however, that there exists one all-encompassing ideal medium or system of culture, and conditions may have to be optimized for each individual cell type or tissue, particularly for human material. Failure to contend with these and similar problems may serve to further perpetuate the myth of culture being inevitably associated with radical changes in the behavior of cells and tissues. A further goal must also be dispensing with the use of serum to sustain cultured cells, as many of the complications that beset endocrine cultures in particular stem from its use. Although efforts in this direction are being made, much remains to be discovered about the precise role played by serum in cultures and the way by which it can be replaced by defined constituents.

I Introduction

II Options in Tissue Culture

A Culture Media

B Culture Systems

III Regulation of Functional Activity and Growth in Endocrine Cells in Culture

A Steroid-Secreting Cells in Culture

B Calcitonin-Secreting Cells in Culture

IV Secondary Applications of Endocrine Cultures

A Hormone Production

B Biological Assay Systems

V Discussion

References

I Introduction

During the last few years there has been a considerable increase in the use of tissue culture in endocrinology as in all fields of biomedical investigation, both as a means of exploring basic endocrine phenomena, and for studying endocrine aspects of cancer. This renewed interest has been largely due to the development of ultrasensitive analytical methods, exemplified by the radioimmunoassay, by which levels of hormones synthesized by individual cultures can now be readily measured. In addition, recent years have seen an increasing availability of a wide range of culture requisites such as defined media, sera, and culture vessels. These factors have greatly increased the practical feasibility of tissue culture, which now provides numerous models for investigating the functional activity of normal and neoplastic endocrine and paraendocrine tissues (Ellison and Neville, 1973).

In its ultimate form, a capability of tissue culture to sustain all tissue-specific functions and responses in vitro for an indefinite period of time under completely defined conditions would allow it to supersede a variety of other methods. At the present time, however, it is clear that there is still some way to go before this goal is achieved. Few, if any, of the tissue culture models of endocrine function currently in use provide a complete facsimile of endocrine cells and tissues in vivo. Nevertheless, the capability to dissect endocrine relationships in vitro affords a powerful tool for their analysis that cannot be ignored. Although problems remain in defining the precise conditions of culture appropriate to specific tissues and functions, it has proved possible by judicious selection of techniques to reproduce certain essential features of endocrine behavior for extended periods of time in vitro. Even when changes in functional activity occur as a result of culture, they can indirectly illuminate the factors that regulate hormone secretion in vivo. A notable example of this was the demonstration of the enhanced secretion of prolactin by cultured hypophyses freed from the inhibitory influence of the hypothalamus (Meites et al., 1961; Pasteels, 1961).

On the whole, however, the early years of endocrine tissue culture were not particularly encouraging. Following the pioneering experiments of Carrel and Burrows (1910), only intermittent attempts were made to culture endocrine glands until the advent of reliable methods of culture with the use of antibiotics to control microbial contamination. Even then its impact was not great because of the relative insen-sitivity of the then current methods of hormone analysis although human chorionic gonadotropin (HCG) secretion in vitro was demonstrated by Gey et al. in 1938. Many cultures, however, failed to produce measurable levels of hormones in spite of the fact that evidence of continued activity could sometimes be inferred from morphological changes seen in co-cultured responsive tissues (for review of early work, see Gaillard and Schaberg, 1965). Nonetheless, tissue culture made some outstanding, if isolated, contributions during this period. These included the identification of somatomedin (sulfation factor) using cultures of cartilage (Salmon and Daughaday, 1957) and later of hypothalamic-releasing factors using pituitary cultures (see Burgus et al., 1976).

At this time, however, the general failure to demonstrate specialized functions in cultures of adult cells led to the widely disseminated assumption that such cells dedifferentiated in culture. A belief therefore grew that tissue culture methods as a whole were implicitly unsuited to a study of endocrine functions, a view that is still held by some today (Schulster et al., 1976). The misapprehension that all adult cells inevitably dedifferentiate in culture has now been dispelled to a large extent by a body of definitive evidence to the contrary (for general reviews, see Green and Todaro, 1967; Wigley, 1975). It has now become clear that most of the unsuccessful early attempts failed for essentially technical reasons such as the overgrowth of cultures by adventitious cell types present in the original tissues, including fibroblast-like cells (Sato et al., 1960) and vascular endothelial cells or pericytes (Franks and Wilson, 1970).

Interest in tissue culture in endocrinology has now revived from the almost universal skepticism of a decade ago to the extent that numerous attempts are now being made to derive significant information from almost all conceivable hormone-producing cells and tissues. It is the purpose of this chapter to outline various modes of tissue culture currently available and applicable to endocrine tissues, illustrated by selected examples drawn from our own experience as well as that of other workers. Pitfalls and problems in the use of tissue culture in endocrinology undoubtedly exist, and by discussing and illustrating some of them we hope to place current progress in this field in a critical perspective. No attempt at a comprehensive review of endocrine cultures will be made, since this would be rapidly outdated at the current rate of progress. Furthermore, in some instances cultures of specific endocrine glands have been recently reviewed in this manner (for the anterior pituitary, see Tixier-Vidal, 1975).

The examples illustrated here will be drawn in the main from cases where tissue culture has afforded the most suitable, and in many cases the only method whereby certain problems could be examined. Emphasis will be placed on human studies since culture techniques here stand paramount as one of the few ethical experimental techniques with which the biology of living normal and pathological human cells can be explored, compared, and contrasted. For reasons of space and uniformity we have limited our consideration primarily to recognized hormone-producing cells of both normal and paraendocrine types (Ellison and Neville, 1973). Many of the problems and pitfalls that emerge from these studies, however, apply with equal force to cultures of hormone-responsive tissues and cells.

In detail, therefore, we will consider the various options in terms of culture methods, and illustrate their inherent advantages and disadvantages as applied to endocrine tissues in relation to (1) regulation of functional activity and growth and (2) the use of endocrine cultures in a secondary context as, for example, hormone factories or assay target tissues.

It is explicitly our intention to attempt, on the basis of these selected examples, to define some of the basic prerequisites for the successful use of tissue culture in endocrine research.

II Options in Tissue Culture

By convention (Fedoroff, 1967), culture begins when cells, tissues, or organs explanted from animals have been maintained in vitro for more than 24 hours. While this distinction from short-term in vitro techniques is an arbitrary one, it is justified by the considerable differences in the complexity of the artificial environment that is provided in order to sustain metabolic activity in vitro over these different time scales.

Methods of tissue culture offer various options—organ culture and cell culture, primary cultures and cell strains, clones and established lines in continuous cultivation. (For definition of terminology, see Fedoroff, 1967; for basic methods, see Kruse and Patterson, 1973.) These options all provide significant alternatives with specific advantages and disadvantages which will be discussed. All, however, require the provision of a complex culture medium, which can play a significant role in determining the behavior of hormone-producing cells and tissues. This medium is usually composed of a chemically defined medium with a buffering system, antibiotics, and serum.

A Culture Media

1 Chemically Defined Media

The primary objective of tissue culture has usually been to sustain viable explanted cells and tissues for as long as possible, and to obtain and enhance proliferation. The criterion of success has therefore been cultures of rapidly dividing cells (in the case of cell culture) or healthy-looking explants (in the case of organ culture), irrespective by and large of their functional activity. This quest has led to the development of a wide array (over 40 basic types in current use) of chemically defined media on a largely empirical basis.

The complexity of these chemically defined media (see Waymouth, 1972) defies simple analysis. One of the most complex, NCTC 109, contains 69 individual components (Evans et al., 1964), whereas one of the simplest, T8, contains a mere 24 (Trowell, 1959). Of relevance in endocrine studies is the fact that some media may contain added hormones (e.g., insulin in T8). Deciding which medium is best suited to an individual cell type or tissue is no mean task, particularly since the specific nutritional requirements of primary cultures in general, and human cells in particular, remain to be established (Ham, 1974). In no case was any readily available medium developed to maintain a specific endocrine tissue (see Waymouth, 1972), and therefore the choice of medium in most endocrine studies has been largely an arbitrary one.

The osmotic pressure of the medium is an example of a potentially uncontrolled parameter that may influence endocrine cells. Thus the tonicity of media may range from 280 to 320 mosmoles/liter (McLimans, 1969), a significant variation in view of the fact that a change of as little as 10% may result in a 50% change in levels of pituitary peptide secretion in vitro (La Bella et al., 1975).

Choosing a medium on a rational basis is complicated not only by the wide differences in basic composition but also by the wide variation in the concentration of individual components. To take just one example, Ca²+, which is intimately involved in many processes of hormone synthesis and secretion, can range from 0.3 mM in Ham’s F-12 to 2 mM in the equally widely used Eagle’s medium (Waymouth, 1972). It is clear, therefore, that many existing medium formulations are not necessarily specifically suited to endocrine tissues, in spite of their ready availability. The concentrations of basic constituents may consequently require changing in accord with the specific requirements of the cells, for example, K+ and aldosterone-secreting cells (Hornsby et al., 1973) or Ca²+ and calcitonin-secreting cells (Roos et al., 1975). In the long run, however, specific media may have to be developed for such specialized cells, preferably ones simpler than the existing formulations, whose complexity is in many cases unsubstantiated by systematic experiment.

2 Buffering Systems

In current tissue culture practice, the use of bicarbonate–CO2 buffering systems to maintain a physiological pH in cultures has to some extent been superseded by the use of the synthetic Good’s buffers (see Eagle, 1971), such as HEPES (4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid). These have reduced or eliminated the requirement for CO2 in the gas phase and with it the necessity for a closed culture system (i.e., stoppered flasks or CO2 incubators). Mammalian cells may, however, require CO2 as a metabolic nutrient (McLimans, 1972). Furthermore, since HEPES buffers well against alkaline changes and bicarbonate–CO2 against acid changes (Shipman, 1976), the best solution for a reproducible, controlled pH in culture is therefore possibly a mixed HEPES–bicarbonate system, or alternatively a more complex mixture of Good’s buffers (Eagle, 1971). Nevertheless, an indiscriminate use of synthetic zwitterionic buffers should be avoided, as should their use at high concentrations, since there is some evidence that they may influence directly the behavior of differentiated cells in culture (Morris, 1971; Daniel and Wolf, 1975; Pfeiffer and Eagle, 1976).

3 Antibiotics

Penicillins and streptomycin have been routinely included in tissue culture medium for so long that their presence passes virtually unnoticed. While no significant deleterious effects have been detected when used at antimicrobial concentrations (up to 100 U/ml and 100 μg/ml, respectively), this innocuous property may not hold for newer antibiotics such as amphotericin B (Dolberg and Bissell, 1974) which may have significant effects on the cells themselves.

In continuously cultivated cell lines there is always a danger of mycoplasma contamination (see Fogh, 1973), which may be difficult to eliminate completely with antibiotics. Covert mycoplasma infections may slow growth and possibly even cause phenotypic alterations in cultured endocrine cells (Schimmer, 1976). Mycoplasmas may also cause chromosomal aberrations in infected cells (Fogh, 1973), a fact that may account for phenotypic changes. Viruses are another insidious contaminant that may change the behavior of cultured cells, and they can often be inadvertently introduced into cultures by serum purportedly free of contamination (Kniazeff et al., 1975). At the present time little can be done to prevent such contamination under normal working conditions, except to screen for such agents whenever a marked change in the behavior of cultured cells is observed. In our experience, however, the newly introduced antibiotic minocycline (Lederle) is particularly effective in controlling mycoplasma infections when used at a concentration of 1 μg/ml.

4 Serum

The greatest source of potential variability in the composition of culture media lies in the serum commonly used to supplement the chemically defined component. Historically, plasma, serum, lymph, and tissue extracts were initially used undiluted for culture. With the development of chemically defined media, however, the requirement was diminished but not, in most cases, completely abolished. Most culture systems in use today contain serum at concentrations between 2 and 20% (v/v).

Serum promotes a variety of functions in cultured cells, including attachment, spreading, migration, nutrient transport, and proliferation (for reviews, see Temin et al., 1972; Sato, 1975). It is therefore particularly important in cell cultures, although less so in organ cultures, some of which can be maintained for limited periods without its use (Trowell, 1959).

In spite of considerable effort, no effective chemically defined substitute for serum has yet been devised, probably because the various functions described above depend on different serum components. Proliferating cell lines can sometimes be adapted to low serum (< 2%) or even a peptone-supplemented medium (Taylor, 1974), and the more hardy lines can be adapted to serum-free medium (e.g., Evans et al., 1964). Attempts to culture endocrine tissues in serum-free medium have not, however, been particularly encouraging to date. Thus, primary cultures of neonatal pancreas have been set up in serum-free medium (Leiter et al., 1974) but the cells failed to respond to the appropriate stimulus for insulin secretion, i.e. glucose, and eventually died. Hayashi and Sato (1976) have recently reported that the GH3 functional rat pituitary cell line will proliferate for some time in serum-free medium supplemented with triiodothyronine (T3), thyrotropin-releasing hormone (TRH), transferrin, parathyroid hormone (PTH), and a partially purified somatomedin. They reported further that the nonendocrine HeLa and BHK lines can be serially propagated in serum-free medium by the addition of 25 hormones. One of the roles of serum in culture medium, therefore, appears to be the provision of hormones, although it is doubtful if this is its only