Biomedical subjects

Richard A Lockshin

Publications and source records attributed to Richard A Lockshin.

6 recordsLinked to original sources

PubMed2005

A generalized caspase inhibitor disrupts early mammalian development.

The role and mechanism of cell death in early mammalian embryos is not well understood. In mouse embryos collected after fertilization and maintained in vitro until blastula formation, two instances of cell death are observed: the polar bodies and one or two cells near the equator, at the junction of the inner cell mass to the prototrophoblast. Inhibitors of caspases do not block the death of the polar bodies. Inhibitors of caspases 3, 7 and 8 do not affect post-cavitation death, but the pan-caspase inhibitor zVAD-FMK, when applied at the 1-2 cell stage, causes an expansion of post-cavitation death and ultimately malformation or death of the embryo. Our results indicate that the early deaths are not caspase-dependent and that there is a role for caspase activity in early embryos, which is not related to cell death.

Amino Acid Chloromethyl Ketones↗

PubMed2004

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.

Animals↗

PubMed2004

Activation of apoptosis and caspase-3 in zebrafish early gastrulae.

Nonmammalian vertebrate embryos do not manifest apoptosis before gastrulation, and it has been suggested that their cells are inhibited from undergoing apoptosis. To study this interesting possibility, the zebrafish (Danio rerio) embryo is an excellent model. However, the appearance of apoptosis varies among species, and many components of cell death are not highly conserved. To undertake the larger investigation, we first need to document by several criteria that cell death in the zebrafish embryo is apoptotic. Exposure of gastrulating germ-ring stage embryos to cycloheximide or staurosporine elicits an arrest in development and cell death within 8 hr. Caspase-3 activity increases, followed by translocation of phosphatidylserine, loss of cell-cell adhesion, cleavage of poly (ADP-ribose) polymerase, terminal deoxynucleotidyl transferase-mediated dNTP-fluorescein nick end labeling (TUNEL)-positive nuclei, internucleosomal DNA fragmentation, chromatin condensation and margination, and blebbing of the nuclear membrane. Thus, by many criteria, cell death in zebrafish is apoptotic; many of the markers of apoptosis found in mammals are conserved in zebrafish; and post-midblastula transition embryos have the capacity to activate a caspase-dependent apoptotic response well before naturally occurring programmed cell death is seen.

Adenosine Diphosphate Ribose↗

PubMed2004

Apoptosis, autophagy, and more.

Cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II has never been completely clear and perhaps does not exist due to intrinsic factors among different cell types and the crosstalk among organelles within each type. Apoptosis can begin with autophagy, autophagy can end with apoptosis, and blockage of caspase activity can cause a cell to default to Type II cell death from Type I. Furthermore, autophagy is a normal physiological process active in both homeostasis (organelle turnover) and atrophy. "Autophagic cell death" may be interpreted as the process of autophagy that, unlike other situations, does not terminate before the cell collapses. Since switching among the alternative pathways to death is relatively common, interpretations based on knockouts or inhibitors, and therapies directed at controlling apoptosis must include these considerations.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

Caspase-independent cell deaths.

A very common and the best understood of the mechanisms of physiological cell death is apoptosis, resulting from the activation, through either of two primary pathways, of site-specific proteases called caspases. There are, however, many other routes to cell death, prominently including autophagy and proteasomal degradation of critical constituents of cells. These routes are frequently seen in experimental situations in which initiator or effector caspases are inhibited or blocked through genetic means, but they are also encountered during normal physiological and pathological processes. Most frequently, autophagic or proteasomal degradation is used to eliminate massive cytoplasm of very large cells, especially post-mitotic cells, and these pathways are prominent even though caspase genes, messages, and pro-enzymes are found in the cells. These forms of cell death are fully physiological and not simply a default pathway for a defective cell; and they are distinct from necrosis. We do not yet understand the extent to which the pathways are linked, what mechanisms trigger the caspase-independent deaths, and how the choices are made.

Animals↗