Biomedical subjects

H Fearnhead

Publications and source records attributed to H Fearnhead.

4 recordsLinked to original sources

PubMed1997

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.

The activity of ICE-like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis-inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.

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PubMed1994

Cdc2 activation is not required for thymocyte apoptosis.

Apoptosis is a mode of cell death characterized by chromatin condensation and disassembly of the nuclear lamina, processes that are also characteristic of mitosis. The apparent similarity between the two events, together with observations that apoptosis can occur following G2 arrest, has led to the suggestion that apoptosis could be a defective form of mitosis. Further support for this idea comes from the recent description of activation of the p34cdc2 protein kinase (Cdc2), the universal M-phase promoter, during death induced by a lymphocyte granule protease. We have monitored the protein kinase activity of Cdc2 during apoptosis of primary rat thymocytes, which die from a quiescent (G0) state. We demonstrate unequivocally that activation of Cdc2 is not involved in the induction of apoptosis in thymocytes, indicating that chromatin condensation and lamina disassembly occur in this system by processes different from those that operate in mitosis.

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PubMed1994

Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes.

Apoptosis is a process in which cells die in a controlled manner and apparently participate in their own demise. It is best characterized morphologically by condensation of chromatin and biochemically by cleavage of chromatin at internucleosomal regions to yield a classical DNA ladder pattern. Apoptosis was induced in thymocytes by exposure to either the glucocorticoid, dexamethasone, or DNA topoisomerase II inhibitor, etoposide. We describe the formation of large m.w. fragments of DNA, 30 to 50 kilobase pairs in length, in a population of these thymocytes at an early stage of apoptosis before internucleosomal cleavage of DNA. These fragments are absent in normal thymocytes and their formation is dependent on protein synthesis. Their appearance is coincident with the commitment of these cells to apoptosis. The formation of these large fragments is associated with the condensation of chromatin, abutting the nuclear membrane, recognized as one of the earliest ultrastructural signs of apoptosis. Subsequent cleavage of these large fragments gives rise to oligonucleosomal fragments and is independent of protein synthesis. We propose that the formation of large fragments of DNA represents a key committed step in apoptosis, and that it is from these fragments that the archetypal DNA ladders associated with apoptosis are derived.

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PubMed1993

Increased membrane permeability of apoptotic thymocytes: a flow cytometric study.

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes.

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