Download a radiation resistant neuroblastoma subline displays reduced

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Biochemical Society Transactions ( 1 996) 24
H37
CHARACTERISATION OF THE BCL-2 INTERACTING PROTEIN, R-RAS.
Alex Eccleston, Maria Jose Fernandez-Sarabia and Frank
McCormick. Onyx Pharmaceuticals, 303 1 Research Drive,
Richmond, CA 94806, USA.
Bcl-2 is a key regulator of apoptosis, but its mechanism of action
remains unknown. Using Bcl-2 as the bait in a two-hybrid
screen, R-ras was isolated and this interaction was confirmed in
vivo. R-ras is a member of the ras family of GTP binding proteins
and shares an identical core effector domain with H-ras. We have
shown that the interaction between Bcl-2 and R-ras is specific
since an interaction was not observed between Bcl-2 and either
H-ras, or TCZl/R-rasE. Although R-ras has been shown t o
interact in vitro with some of the same effector molecules as
H-ras, i t appears that R-ras has a different biological role t o
H-ras. For example, whilst oncogenic H-ras can transform rat-1
cells, activated R-ras (V38) cannot. To investigate if R-ras can
activate the same signalling pathways as H-ras, we used activation
of the MAP kinase cascade in cos cells as a read-out. Whilst H-ras
efficiently activates c-Raf and the MAP kinase, Erk-2, R-ras does
not. R-ras is localised t o the plasma membrane in these cells, as
is H-ras, but appears less efficient than H-ras in translocating
c-Raf there. Chimaeras between H-ras and R-ras have been made
and their kinase activity is being tested t o distinguish which
structural features of R-ras determine its differential behaviour
t o H-ras. These chimaeras are also being tested in an IL-3
dependent B cell line, t o elucidate the mechanism of the previously
observed effect of R-ras in suppressing Bcl-2 action in these
cells.
H39 EXPRESSION OF APOPTOSIS-MODULATINGGENES IN
HUMAN MULTIDRUG RESISTANT CELL LINES
L Connolly, R NicAmhlaoibh, S Verhaegen, and M Clynes.
National Cell and Tissue Culture Centre, Dublin City University,
Dublin, Eire.
The expression of apoptosis-modulating genes was investigated in
sensitive and multidrug resistant variants of two human cell models, the
lung carcinoma cell line DLKP (1) and the myelomonocytic cell line HL60, the latter a well studied model for apoptosis (2).
In all of the cell lines tested mRNA for the bcl-2, bcl-xL, bcl-a, and bax
genes could be detected using RT-PCR. Large differences between the
sensitive and resistant variant were not apparent, except in the case of
the apoptosis-suppressinggene bcl-xL, which was increased 2 to 3-fold
in the resistant variant, DLKP-A. Bcl-2 and bax were also examined at
the protein level using Westem blot. Bcl-2 protein was not detected in
DLKP or its variant, but was present in both the sensitive HL-60s and
the multidrug resistant variant HL-6OADR. The apoptosis-promoting
gene, bax was expressed in all the DLKP-variants, and in HL-GOS, but
was absent in HL-GOADR.
From time-lapse videomicroscopy observations we know that an
apoptotic pathway is intrinsically available in both the sensitive parental
line, DLKP and the P-glycoprotein expressing variant, DLKP-A.
Similarly, morphological analysis of both HL-60s and the MRP
expressing variant HL-6OADR show the intrinsic capability of cell death
via apoptosis when treated with a wide variety of anti-cancer drugs.
These results indicate that, in addition to overexpressing protein
involved in drug efflux (e.g. P-glycoprotein or MRP). rnultidrug resistant
cells may show altered expression of proteins which determine
sensitivity of the cell to drug-induced apoptosis.
Clynes M, Redmond A, Moran E, and Gilvany U. Multiple
(1)
Drug-resistance in a variant of non-small cell lung carcinoma cell line,
DLKP-A. Cytotechnology. 70: 75-89 (1992).
(2)
Cotter TG, Femandes RS, Verhaegen S, and McCarthy JV.
Cell death in the myeloid lineage. Immunol. Rev., 142: 93-112 (1994).
H40 A RADIATION RESISTANT NEUROBLASTOMA
H38 INCREASED LEVELS OF BCL-XLEXPRESSION
CORRELATE WITH RESCUE FROM APOPTOSIS IN
THE BURKITTS-LYMPHOMA CELL LINE RAMOS.
and Kirstine b o x , Department o f Biochemistry,
University o f Oxford, South Parks Rd, Oxford, OX1 3QU.
Introduction: Selection via inhibition o f a default apoptotic
pathway plays a central role during development o f the immune
response. One such selection pathway occurs in the germinal
centre (GC) during B cell affinity maturation. The Ramos-BL B
cell line originates from transformation o f GC B cells and thus
retains a vestige o f GC selection: apoptosis induced by ligation
o f surface immunoglogulin (sIgM) can be rescued by signals
through CD40. In GC cells rescue i s mediated, in part, by Bcl-2
up-regulation. Aim: The potential role o f Bcl-2 and its related
proteins Bax and Bcl-x, were investigated during induction and
suppression o f apoptosis in Ramos-BL B cells. Results: Bcl-x,
was found to be expressed as a doublet o f 29 kDa and 31 kDa:
the 31 kDa isoform localises to the cytosol whereas the 29 kDa
form i s membrane-bound. Rescue signals were shown to be
accompanied by an increase in the expression o f both Bcl-2, by
48h, and Bcl-xL-p29, by 12h post-treatment. Levels o f Bcl-xLp3 1 and Bax were unchanged. Conclusion: Our results indicate
that CD40-induced survival may be initially mediated by Bclx,-p29 and only later by Bcl-2. This suggests a role for Bcl-x,p29 in offering transient protection from apoptosis in GC cells
undergoing selection.
SUBLINE DISPLAYS REDUCED APOPTOSIS AND
REDUCED LEVELS OF BCL 2
James Russell, The Johns Hopkins Oncology Center, 600 N Wolfe
St, Baltimore, MD 2 1287 USA
A population of radiosensitive human neuroblastoma IMR32 cells
was exposed to repeated doses of 2 Gy X-irradiation, resulting in
the emergence after 15 such fractions of a more resistant
population. The fraction of cells surviving a dose of 2 Gy was 0.05
for the parental cells as opposed to 0.19 for the resistant cells.
Although these cells were unstable and tended to revert to a parental
phenotype, it was possible to clone out cells which maintained the
resistant phenotype over 35 passages. The resistant and parental
cells did not differ in their ability to rejoin radiation-induceddouble
strand breaks, and both lines displayed similar levels of
chromosome damage, as judged by micronucleus frequency &r
radiation. However, the resistant cells showed significantly less
apoptosis following irradiation, implying that the reduced
susceptibility to radiation-inducedapoptosis was responsible for the
enhanced clonogenic survival.
The molecular basis for these differences is being pursued.
The cells do not differ in their p53 status, which i s wild-type in both
lines. With regard to the Bcl-2 family, the results are paradoxical.
Bcl-2 i s more highly expressed in parental cells than in the resistant
line. In neither line does expression change following mhation.
Constitutive levels of Bax are approximately the same in both cell
lines, though the Bax protein level falls after radiation exposure, but
only in the parental cells. Bcl. expression i s similar in parental and
resistant cells. Bcl. i s not expressed in either line. The results
suggest that other molecules are duencing apoptosis in these cells.