Download analysis of proteins that bind to bcl

600s Biochemical Society Transactions ( 1 996) 24
H29 USE OF COMPETITIVE PCR TO MONITOR
EXPRESSION OF THE bcl-2, bcl-x, mcl-I, bw,bak
AND bf-I GENES DURING THE INDUCTION OF
APOPTOSIS IN HUMAN HXMATOPOIETIC CELL
LINES
ChristoDher A. Smith, Sue K. Ganderton, Aedin C. Culhane,
Pedro S. Leite and Gwyn T. Williams
Department of Biological Sciences, Keele University,
Staffordshire ST5 5BG, UK
We have developed a competitive PCR system to follow
changes in the relative abundance of the major mRNAs encoding
Bcl-2, Bcl-xL, Bcl-Q, Mcl-I, Bax, Bak and Bfl-1. These proteins
are all members of the family of human proteins related to the
Cmorhabditis eleguns apoptosis-regulatingprotein Ced-9. Bcl2 and Bcl-x~(and probably also Mcl-1 and Bfl-1) inhibit or delay
apoptosis, whereas Bax,Bak and Bcl-xs promote apoptosis
Using this system we have monitored changes in the expression
of these genes in three human hrematopoietic cell lines following
treatments that induce cell death by apoptosis.
The human cell lines used were Jurkat (T-cell leukamia), HL60
(promyelocytic leukremia) and TF1 (IL-3-dependent erythroleukaemia).
The treatments used to induce apoptosis were X-irradiation,
treatment with the topoisomerase 11-inhibiting chemotherapeutic
agent etoposide (VP-16) and cytokine deprivation (IL-3
withdrawal).
H30
ANALYSIS OF PROTEINS THAT BIND TO
BCL-2 DURING APOPTOTIC STRESSES
H31 TOXICITY OR NON-TOXICITY OF SPHINGOMYELIN/N-HEXANE/ETHANOL DISPERSIONS DEPENDS
ON THE MODUS OF ITS PREPARATION
Svbille G. E. MevQrl, Nicole Cariusl , Corinna Bruhnkel ,
Karin Flumannl, Anton G. Rietvelde and Herbert de Grootl
1lnstitut fur Physiologische Chemie, Universitatsklinikum
Essen, Hufelandstr. 55, D-45147 Essen, Germany and
2Centre for Biomembranes and Lipid Enzymology,
Department of Biochemistry of Membranes, University of
Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
Bovine brain sphingomyelin and other long-chain
phospholipids dissolved in n-hexanehthanol (1 :1, v/v) and
injected into the incubation medium to give a final solvent
concentration of 0.4 YO was highly toxic for the tumour cell
line L929 and other cells. In contrast, the short-chain
phosphatidylcholine (C4) was non-toxic. Sphingomyelin
dispersions with 0.08 YO solvent were always non-toxic and
stayed still non-toxic even if the solvent was adjusted to 0.4
or 2 Yo.Quite the opposite was true if sphingomyelin
dispersions were prepared with 2 Yo solvent from the start.
These preparationswere always toxic.
Fluorescent sphingomyelin was preferentially taken up by
the plasma membrane. Cell death was preceded by an
increase in the cytosolic Ca2+ concentration. 31P NMR
spectra of the sphingomyelin/solvent dispersions showed
isotropic and bilayer components.
The results suggest that toxicity is caused by the integration
of hexane-loaded phospholipids into the plasma membrane
inducing membrane damage. Non-toxic lipid aggregates
presumably are microemulsions which enable the
phospholipid to be integrated into the plasma membrane
without hexane.
H32 MODULATION OF APOPTOSIS BY INHIBITION
OF CED-3nCE LIKE PROTEASES
McC-.
Moira Whyte and Gerard Evan
Biockmistry of the Cell Nucleus laboratory, 44 Lincoln’s Inn Fields, London.
w a 3PX
Studies of programmed cell death in both mammalian and invertebrate model
Otter. I., and Borner, C.
systems have successfully identified two conserved gene families involved in the
regulation of apoptosis: the ced-9/bcl-2 family and the ced-jlice family. For the
Institute of Biochemistry, University of Fribourg, 1700
Fribourg
The oncogene product Bcl-2 effectively spares mammalian
cells from programmed cell death induced by numerous
stimuli. The molecular basis for this death-protectiveactivity
is still a matter of debate. In order to find partners of Bcl-2
mediating its action, we devised an immunoprecipitation
approach to identify proteins which specifically associate
with Bcl-2 under physiological and non-physiological
apoptotic stresses. Here we present a well-known
antagonist of Bcl-2, called Bax whose interaction with Bcl-2
remains intact during apoptotic stresses. In addition, a novel
protein is shown which appears to co-precipitate with Bcl-2
only under stress conditions. The identification of the latter
protein may pave the way to uncover the impatiently awaited
molecular function of Bcl-2.
Reproduced with permission from Expenentia 52, A79 (1996).
more recently characterised interleukin-I B-converting enzyme (ICE) family of
cystcine proteases there is increasing evidence for a central role in mammalian
apoptosis. Whether these enzymes are part of the basal machinery of apoptosis or
part of its regulation, like members of the bcl-2 family, is uncertain, as is the
absolute requirement for one or more of these proteases for completion of the
apopotic process.
Our studies employ 2-Val-Ala-Asp. fluoromethylketone (ZVADfmk), a cell
permcable inhibitor of some of these proteases and demonstrate: (1) That ICE-like
protases are involved in apoptosis induced by disparate mechanisms (deregulated cMyc expression, Bak overexpression, adenovirus EIA expression, or p53 mediated
etoposide-induced DNA damage). (2) That ZVADfmk treatment does not alter h e
time &en to onset of apoptosis. unlike Bcl-2. Instead ZVADfmk delays the time
takato complete the cellular changes of apoptosis. thus assigning active ICE-like
p r o w to a role in the basal machinery of apoptosis rather than its regulation.
(3) m a t ZVADfmk inhibits the characteristic biochemical and morphological
evens associated with apoptosis. including chromatin condensation, DNA
laddaing. Annexin V binding and cleavage of nuclear lamins and polyADP-ribose
polyrerase ( P A W .