Richard A. Lockshin

Cell death in biology and pathology

This book should be of interest to students and researchers in biochemistry and cell biology.

Caspase-independent cell death?

Many cells die with apoptotic morphology and with documented activation of an effector caspase, but there are also many exceptions. Cells frequently display activation of other proteases, including granzymes, lysosomal cathepsins, matrix metalloproteinases, and proteasomal proteases, and others display morphologies that are not fully consistent with classical apoptosis. In some experimental situations, evidence of caspase-dependent death is indirect, demonstrating that the cell can activate caspases rather than that it does. In other situations, such as involution of mammary or prostate tissue, many cells display autophagic or other morphology different from apoptosis, and there is considerable evidence for the activation of a lysosomal system. Prior to total collapse and necrosis, cells that are in trouble can activate numerous physiological pathways toward self-destruction. Intrinsic or extrinsic routes to effector caspase activation are frequently the most rapid and efficient. If neither of these routes is immediately available, owing to mutation, genetic manipulation, inhibitor, or the biology of the cell, other routes may be followed, leading to variant forms of cell death that may display one or more characteristics of apoptosis. Experimental and therapeutic procedures must account for this possibility.

Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein.

Abstract The death of the intersegmental muscle of saturniid silkmoths is controlled by a complex series of events involving the endocrine and neural system. The signal to initiate lysis is produced anterior to the abdomen. The actual initiation of the lytic phase can be blocked by puromycin, actinomycin D, or cycloheximide at levels consonant with their ability to inhibit synthesis of proteins and RNA. The action of the antibiotics is manifested in an isolated denervated abdomen. The activity of the lytic enzymes already present in the muscle is not significantly inhibited by either puromycin or actinomycin D. The data suggest that the lysosomes present in the muscle are activated by a process involving the synthesis of RNA and protein.

Cell death during development

There are many ways to measure apoptosis and other forms of programmed cell death in development. Once nonmammalian embryos have passed the midblastula transition, or much earlier in mammalian embryos, apoptosis is similar to that seen in adult organisms, and is used to sculpt the animal, fuse bilateral tissues, and establish the structure of the nervous system and the immune system. Embryos present unique problems in that, in naturally occurring cell deaths, few cells are involved and they are frequently in very restricted regions. Thus, identification of apoptotic or other dying cells is more effectively achieved by microscopy-based techniques than by electrophoretic or cell-sorting techniques. Since embryos have many mitotic cells and are frequently more difficult to fix than adult tissues, it is best to confirm interpretations by the use of two or more independent techniques. Although natural embryonic deaths are frequently programmed and require protein synthesis, activation of a cell death pathway is often post-translational and assays for transcriptional or translational changes-as opposed to changes in aggregation of death-related molecules or proteolytic activation of enzymes-is likely to be uninformative. Also, embryos can frequently exploit partially redundant pathways, such that the phenotype of a knockout or upregulated death-related gene is often rather modest, even though the adult may develop response or regulation problems. For these reasons, the study of cell death in embryos is fascinating but researchers should be cautious in their analyses.

Cell death in metamorphosis

Metamorphosing animals provide the most obvious models for the study of cell death. In insects and amphibia, large masses of relatively homogeneous tissue degenerate rapidly and predictably. In many instances, the extracellular controls are well understood and the control manipulatable at the experimenter’s convenience; the response may be evocable in vitro. In other instances of rapid development—the metamorphosis of embryonic invertebrates (molluscs, echinoderms or ascidians) or tissue metamorphosis (Mullerian and Wolffian ducts, ovulation, involution of the corpus luteum) — there are equal opportunities for experimentation, but they have not been as thoroughly studied (Glucksmann, 1951).

Insect embryogenesis: macromolecular syntheses during early development.

A new technique permits the injection of aqueous solutions into the eggs of certain Coleopteran insects. DNA and protein are synthesized from the outset of development, but the synthesis of RNA is not detectable until the ligrating cleavage nuclei arrive at the cortex of the egg.

Control of ecdysis by heat in Manduca sexta

Under constant-light conditions, adult Manduca sexta emerge approximately 5 hr after the middle of a 3 hr 3°C heat pulse. The insects can be entrained to a heat cycle in 3 days. Entrainment will decrease at approximately the same rate. Photic signals can modify the precision of the response but, when the insects are presented with conflicting photic and thermal signals, they respond to the thermal signal. During the 3 days of entrainment, the insects appear to hasten the time of eclosion as much as 6 hr to meet the thermal cycle, but the degeneration of the intersegmental muscles is not similarly hastened. The technique described can be applied to various physiological and behavioural studies.

Breakdown of the silk glands of Galleria mellonella—Acid phosphatase in involuting glands

Abstract The silk glands of larvae of Galleria mellonella and Malacosoma americana contain sedimentable enzymes digesting paranitrophenyl phosphate, sodium-β-glycerophosphate, and naphthol phosphates at pH 5·0. These enzymes increase significantly in amount, but do not alter in distribution, as the larva pupates.

Degeneration of insect intersegmental muscles: Electrophysiological studies of populations of fibres

Abstract Several parameters were observed during the degeneration of the intersegmental muscles of moths. Within 5 hr after the emergence of the moth, capacitance of the fibres decreases 70 per cent, and resistance begins to climb, the latter reaching a maximum value approximately 300 per cent that at the time of emergence, at 20 hr. The loss in muscle mass is detectable approximately 3 hr after emergence, but the muscle remains fully contractile for at least 10 hr; and intracellular depolarization can elicit localized contractions for another 5 hr. Motor end plates remain potentially capable of function throughout this period. The changes in membrane properties represent the earliest known physiological manifestations of lysis.

Programmed cell death: Nature of the nervous signal controlling breakdown of intersegmental muscles

Abstract The drugs physostigmine, caffeine, and barium chloride, acting through the central nervous system, prevent the breakdown of the intersegmental muscles in silkmoths. Tetrodotoxin and the venom of the wasp, Microbracon hebetor , advance degeneration minimally, if at all; but both inhibitory drugs overcome the protective effect of either physostigmine or barium. No evidence is available to suggest the mediation of other than classical neural activity as a means of controlling the fate of the muscle.