Bariloche protein symposium argentine society for biochemistry and molecular biology

55
BIOCELL, 27 (Suppl. I), 2003
BC-C12.
POSSIBLE MECHANISMS INVOLVED IN c-FOS
ACTIVATION OF PHOSPHOLIPID SYNTHESIS
MM Portal, GA Gil, ML Renner and BL Caputto.
CIQUIBIC, Departamento de Química Biológica, Facultad de
Ciencias Químicas, U.N.C Córdoba, Argentina. E-mail:
[e-mail]
Tumorigenesis is a multi-step process, reflecting complex genome
changes that progressively drive transformation of normal cells
into malignant derivatives. Nuclear c-Fos participates, as a
component of AP-1 transcription factors, in events of cell growth
and differentiation. It´s expression is subjected to tight control
checkpoints, and over-expression can lead to cellular
transformation. We examined the involvement of c-Fos in
tumorigenesis in T98G (glioblastoma multiforme) cell line. We
found that c-Fos associates to the ER, activating phospholipid
synthesis, main constituents of biological membranes. Blocking
c-Fos expression using antisense strategies, inhibits c-Fos
dependent phospholipid synthesis activation, and also diminishes
cell proliferation. In quiescent cells, the small amount of c-Fos
present is phosphorylated in tyrosine residues. Upon stimulation
with trophic factors, this state is rapidly lost and de-phosphorylated
c-Fos is now able to associate to the ER, thus activating
phospholipid synthesis. If this process is impaired by addition to
the culture media of an inhibitor of tyrosine phosphatases, c-Fos
dependent phospholipid synthesis activation drops down and also
does so the proliferation velocity. Interestingly, we found high
levels of c-Fos present for at least 18 hours post-stimulation
indicating that the stability of c-Fos protein or that of its mRNA
could account for its accumulation in these cells. We postulate
that this cytoplasmic activity of c-Fos is a key event in cancer
progression.
Supported by FONCyT, SeCyT-UNC, Agencia Cordoba Ciencias
and JS McDonnell Foundation.
BC-C14.
NUCLEO-CYTOPLASMIC LOCALIZATION OF P8, CELL
CYCLE AND SIGNAL TRANSDUCTION PATHWAYS
P. Valacco, C. Varone, J. Iovanna, and S. Moreno.
Depto. de Química Biológica, FCEN, UBA. E-mail:
[e-mail]
p8, an 8 kDa protein was first identified by its induction during
the acute phase of pancreatitis. Its mRNA is expressed in cell
lines in response to stress and mitogenic factors. With immuno-
cytochemistry, we found that p8 presents nuclear localization in
sub-confluent cells, but it localizes in the cytoplasm of confluent
cells. Further experiments have shown that nuclear import is energy
dependent and that the export of p8 does not involve the CRM1
transporter. Different experiments with cycloheximide suggest that
the protein found in the cytoplasm is the result of a de novo protein
synthesis, and not of the export of nuclear p8. The objective of
this work is to study the sub-cellular localization of the protein
along the stages of the cell cycle with immunocytochemistry.
Different strategies, such as serum deprivation, hydroxiurea,
nocodazole and etoposide were used to arrest cells in the different
stages, and it was observed that in sub-confluent cells, arrested in
Go, the protein localized in the cytoplasm, and when the cell cycle
was restored, p8 migrated back to the nucleus. Since previous
results show that inhibitors of MAPK pathways alter the level of
p8 expression, their effect on p8 localization was assayed, but
none of the drugs had any effect on the protein’s localization. In
the presence of a histone deacetylase inhibitor, Trichostatin A,
sub-confluent cells showed cytoplasmic localization of p8.
TS-C1.
ISOLATION OF THE STYLE PARTNER OF POLLEN
RECEPTOR KINASES LePRK1 AND LePRK2 FROM
TOMATO (LYCOPERSICON ESCULENTUM)
Wengier, Diego; Cabanas, María; Salem, Tamara; Sanchez,
Sabrina; de Paz Sierra, Pablo and Muschietti, Jorge.
INGEBI-UBA-CONICET, Obligado 2490, Ciudad de Buenos Aires.
E-mail: [e-mail]
After pollen grains germinate on the stigma, pollen tubes traverse
the extracellular matrix of the style on their way to the ovules. We
characterized two pollen-specific receptor kinases, LePRK1 and
LePRK2, that according to their structure, immunolocalization
pattern and style extract-mediated specific
 
LePRK2-
dephosphorylation, they might
 
interact with signaling molecules
within the style extracellular
 
matrix. Recently, we demonstrated
that in pollen membranes, LePRK1 and LePRK2 belong to a high
molecular complex of ~400 kDa (Wengier et al, PNAS, Vol 100,
no. 11, pp. 6860-6865, 2003). This complex is disrupted when
pollen is germinated in vitro in the presence of style extracts. In
yeast, the addition of style extract also disrupts
 
the interaction
between LePRK1 and LePRK2. Crude fractionation of
 
the style
extract shows that this activity is enriched
 
in the 3- to 10 kDa.
fraction. From FPLC fractionation, we obtained a broad peak of
~6 kDa. specifically associated with LePRK2 dephosphorylation.
The style component that dephosphorylates LePRK2 is likely
 
to
be a heat-stable peptide that is present in the style exudate. Further
characterization will be necessary to identify the structure of
LePRK ligand.
BC-C13.
CYTOPLASMIC c-FOS: A NOVEL TARGET FOR CANCER
THERAPY?
Silvestre D, Gil GA, and Caputto BL.
CIQUIBIC-Dpto Quimica Biologica, Facultad de Ciencias
Quimicas, Universidad Nacional de Cordoba, Argentina. E-mail:
[e-mail]
Given our results in PC12 and brain tumor cells, we propose that
AP-1-c-Fos is not the critical player in the tumor growth process.
If our hypothesis is correct, cytoplasmic c-Fos should be found in
actively growing cancer cells. A screening for c-Fos expression in
malignant human tumor specimens was performed. Specimens
included: testicular (n=7), lung (n=7) breast(n=10) and liver (n=9)
tumors. Cytoplasmic c-Fos immunoreactivity was observed in
100% of the tumors and all tumors examined showed ER/c-Fos
co-localization. c-Fos expression, phospholipid synthesis activation
and proliferation were examined in the breast tumor cells MCF7
and T47D. Proliferating cells showed c-Fos-dependent activated
phospholipid synthesis whereas cells fed a c-Fos antisense
oligonucleotide showed no phospholipid synthesis activation and
no proliferation.
BalB c/c nude mice were inoculated intracerebrally with T98G
cells to promote tumor growth. At day 7 animals were separated
into 3 groups and were injected at inoculation site, with c-Fos
mRNA antisense oligonucleotide or sense oligonucleotide or
vehicle every 5 days during 20 days. Brain tissue samples were
examined at day 40 for tumor presence. Tumors developed in 6
out of 7 vehicle-, in 3 out of 4 sense-, and in none out of 5 antisense
oligonucleotide- injected animals. These results indicate a relevant
role of cytoplasmic c-Fos in the growth process of tumor cells.
Supported by: FONCyT, SeCyT-UNC, Agencia Cordoba Ciencias,
CONICET, JS McDonnell Foundation.

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