Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

Cell death : the significance of apoptosis

It is proposed that most neoplasms arise from a single cell of origin, and tumor progression results from acquired genetic variability within the original clone allowing sequential selection of more aggressive sublines. Tumor cell populations are apparently more genetically unstable than normal cells, perhaps from activation of specific gene loci in the neoplasm, continued presence of carcinogen, or even nutritional deficiencies within the tumor. The acquired genetic insta0ility and associated selection process, most readily recognized cytogenetically, results in advanced human malignancies being highly individual karyotypically and biologically. Hence, each patient's cancer may require individual specific therapy, and even this may be thwarted by emergence of a genetically variant subline resistant to the treatment. More research should be directed toward understanding and controlling the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.

http://science.sciencemag.org/content/sci/194/4260/23.full.pdf

The clonal evolution of tumor cell populations

Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver cells.

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.

Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis.

https://cancerres.aacrjournals.org/content/canres/44/10/4217.full.pdf

The Multistep Nature of Cancer Development

The sequence of possible cellular, tissue, biochemical, and molecular changes that are important during the development of experimental liver cancer with chemicals is reviewed from the viewpoint of the author's experience in carcinogenesis over the past 25 years. The development of a new model for the analysis of liver carcinogenesis, the resistant hepatocyte model, is briefly described, as are the known steps between exposure to an initiating dose of a chemical carcinogen to the appearance of hepatocellular carcinoma. These steps include: (a) the interactions with DNA; (b) the dependence on a round of cell proliferation for initiation; (c) one type of initiated hepatocyte, a resistant hepatocyte; (d) the selection of these new hepatocytes, probably by clonal expansion, to form synchronously the first type of hepatocyte nodules, early nodules; (e) the election of the majority of these nodules to undergo remodeling to normal-appearing liver by differentiation ("redifferentiation"); (f) the election of a minority of early nodules to persist; (g) the slow growth of the few persistent nodules; and (h) the precursor role of persistent nodules in the development of hepatocellular carcinoma. The evidence for such a precursor role includes: (a) the common occurrence in persistent nodules of a subsequent precancerous step, "nodules in nodules"; (b) the occurrence of metastasizing cancer inside nodules without cancer elsewhere in the liver; and (c) the high rate of evolution to cancer of persistent nodules, but not of early nodules, when transplanted to the spleen. Based on the common architecture, organization, blood supply, and biochemical pattern of properties relating to the metabolism of xenobiotics in hepatocyte nodules in six different models of liver carcinogenesis and on the common occurrence of a highly programmed redifferentiation pattern of carcinogen-induced hepatocyte nodules, it is concluded that heterogeneity and diversity seen in many phenotypic properties of cancers, including liver cancers, is preceded by a precursor population that is unusually homogeneous and uniform in phenotype. Thus, the heterogeneity and diversity of cancers are probably late manifestations in carcinogenesis. The available evidence is very suggestive that the hepatocyte nodules are an expression of physiological adaptation to exposure to hazardous xenobiotics and not a form of aberration or mutation. The data also suggest that hepatocyte nodules are an additional pattern of liver differentiation and that liver cancer, to be understood, should be compared with this precursor new state rather than the conventional adult, fetal, or embryonic states.(ABSTRACT TRUNCATED AT 400 WORDS)

https://cancerres.aacrjournals.org/content/canres/44/12_Part_1/5463.full.pdf

Cellular Biochemistry of the Stepwise Development of Cancer with Chemicals: G. H. A. Clowes Memorial Lecture

The induction of resistant hepatocytes in vivo in the rat has been observed with 21 different chemical carcinogens. The resistance was measured by the ability of the cells to proliferate as focal lesions in the presence of an environment that inhibits the original or surrounding hepatocytes from proliferation. This was created by a dietary 2-acetylaminofluorene plus a stimulus for cell proliferation, a single necrogenic dose of CCl4. The foci were readily visualized by staining for gamma-glutamyl transpeptidase activity. With most of the chemicals, a single administration at an appropriate time after partial hepatectomy is efficacious. However, with safrole and dieldrin, three doses over a 36-hr period were required to induce a significant number of foci of resistant hepatocytes. The presumptive preneoplastic nature of the resistant hepatocytes and the possible usefulness of this approach for the development of a new in vivo short-term test system for carcinogens are discussed.

https://cancerres.aacrjournals.org/content/canres/40/4/1157.full.pdf

Induction of Resistant Hepatocytes as a New Principle for a Possible Short-Term in vivo Test for Carcinogens

Summary A dietary regimen is described whereby large hyperplastic nodules can be obtained in over half the rats fed either of the two hepatocarcinogens, ethionine or 2-fluorenylacetamide. The nodules are composed predominantly of hepatocytes which show morphologic and biochemical differences from the surrounding nonhyperplastic liver. The glycogen or its metabolic control are different in the nodule in that a significant amount of glycogen is present even after a 48-hour period of fasting and little breakdown is induced by glucagon administration. The nodules show a consistent and progressive decrease in glucose-6-phosphatase activity as well as in glycogen phosphorylase activity. Evidence implicating the hyperplastic nodule as a step in the carcinogenic process is presented and discussed.

https://cancerres.aacrjournals.org/content/canres/27/9/1702.full.pdf

Cellular Analysis of Liver Carcinogenesis: the Induction of Large Hyperplastic Nodules in the Liver with 2-Fluorenylacetamide or Ethionine and Some Aspects of Their Morphology and Glycogen Metabolism

A system is described for the detailed study of the remodeling of hepatic nodules that appear regularly during liver carcinogenesis with chemicals. With the use of the resistant hepatocyte model and by focusing on the caudate lobe, it has been possible to label with [3H]thymidine all the hepatocytes in hepatocyte nodules without any significant degree of labeling of the surrounding hepatocytes. Through such a model, the persistence of the label, in relation to the organization and appearance of the hepatocytes in the nodules, has been followed for 26 weeks. Nodules do not “disappear” to any significant degree by regression or by replacement with hepatocytes from the surrounding liver. Rather, nodule hepatocytes undergo differentiation to an adult liver phenotype. Thus, differentiation (“redifferentiation”) of a carcinogen-induced altered hepatocyte population is seen regularly during carcinogenesis despite the irreversible nature of some of the changes induced by a chemical carcinogen during initiation.

Emmanuel Farber

Papers

The Multistep Nature of Cancer Development

Cellular Biochemistry of the Stepwise Development of Cancer with Chemicals: G. H. A. Clowes Memorial Lecture

Induction of Resistant Hepatocytes as a New Principle for a Possible Short-Term in vivo Test for Carcinogens

Cellular Analysis of Liver Carcinogenesis: the Induction of Large Hyperplastic Nodules in the Liver with 2-Fluorenylacetamide or Ethionine and Some Aspects of Their Morphology and Glycogen Metabolism

Redifferentiation as a Basis for Remodeling of Carcinogen-induced Hepatocyte Nodules to Normal Appearing Liver