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Original Studies |
Centro di Endocrinologia ed Oncologia Sperimentale, Comitato Nazionale Biotecnologie e Biologia Molecolare (G.R.), and Dipartimento di Biologia e Patologia Cellulare e Molecolare (M.V., T.D.M., M.I., G.R.) and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica (G.F.F.), Università Federico II, Naples 80131, Italy
Address all correspondence and requests for reprints to: Dr. Mario Vitale, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini, 5 Naples 80131, Italy. E-mail: mavitale{at}cdsunina.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis or programmed cell death is an active process of
self-destruction that requires the activation of a genetic program
leading to changes in morphology, DNA fragmentation by an endogenous
deoxyribonuclease, and protein cross-linking (3, 4). Although the
molecular mechanisms behind apoptosis remain poorly understood, some
molecular effectors have been identified. It is now clear that the
apoptotic pathway can initiate at the cell surface from membrane
receptors such as Fas/APO1 and tumor necrosis factor receptor-1, and
that cysteine proteases represent one of the effector components of the
apoptotic machinery. Activation of the apoptotic pathway is under the
control of physiological stimuli such as environmental signals,
cytokines (5, 6, 7), and growth factors and can also be induced by
pathological stimuli, radiation, and anticancer drugs (2, 8, 9).
Hormone depletion determines apoptosis in a number of hormone-dependent
tissues, such as prostate and mammary glands (10, 11) or uterine
epithelium. Also, serum withdrawal in endothelial cells as well as in
canine thyroid primary cultures and Kirsten-ras-transformed
rat thyroid cells induces programmed cell death (12, 13). Like hormones
and growth factors, the ECM affects cell behavior and plays an
important role in the regulation of many biological processes,
including cell morphology, differentiation, transformation, and growth
(14, 15). Recent studies demonstrate that in addition to regulating
cell growth and differentiation, ECM is also a survival factor for many
cell types. Most normal cells require attachment to ECM to survive,
whereas the anchorage dependence is reduced or totally absent in
transformed cells. Cell adhesion to ECM is mainly mediated by the
integrins, a family of cell surface receptors widely expressed on all
tissues. The 
ß integrin complex has an extracellular domain
bearing the ligand-binding site and an intracellular domain interacting
with cytoskeletal proteins (16). Some of these receptors colocalize
with several regulatory proteins, such as
pp60src, pp125FAK, protein
kinase C, and mitogen-activated protein kinase, in a subcellular
structure defined as focal adhesion (17). From these subcellular sites
initiates the signal transduction pathway triggered by the integrin-ECM
interaction that contributes to the regulation of many biological
processes, including differentiation, transformation, and growth (14, 18). We previously showed that integrin activation by ECM regulates
cytoskeletal organization and stimulates the proliferation of normal
human thyroid cells in culture (19). In the present study we
demonstrate that the immortalized thyroid cell line TAD-2 is a good
model to study thyroid cell-ECM interaction. TAD-2 cells stimulated by
serum deposit FN as insoluble matrix required for cytoskeletal
organization and to prevent apoptosis through integrin-mediated
adhesion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TAD-2 cell line, obtained by simian virus 40 infection of human fetal thyroid cells (20), was donated by Dr. T. F. Davies, Mount Sinai Hospital (New York, NY), and cultured in a 5% CO2 atmosphere at 37 C in RPMI medium supplemented with 10% FCS. Medium was changed every 3–4 days. Cells were detached by 0.5 mmol/L ethylenediamine tetraacetate in calcium- and magnesium-free phosphate-buffered saline (PBS) with 0.05% trypsin.
Antibodies, immunofluorescent localization, and flow cytometric analysis
For intracellular immunofluorescence (cytokeratin and thyroglobulin), cells were fixed in 3.5% paraformaldehyde, 0.2% Tween-20 in PBS, washed twice in PBS, and resuspended in 0.5% BSA-PBS; immunostaining was then performed using fluorescein-conjugated anticytokeratin antibodies (Ortho, Raritan, NJ) or rabbit antihuman thyroglobulin serum followed by sheep antirabbit IgG as a fluorescein-conjugated secondary antibody. Serum from nonimmunized rabbits or nonspecific fluoresceinated Igs of the same isotype were used as controls. Cells were then analyzed by flow cytometry using a FACScan apparatus (Becton Dickinson Co., Mountain View, CA).
Monoclonal antibodies of mouse origin against the various integrin
subunits were donated as indicated: A1A5 (anti-ß1), Dr.
M. E. Hemler (Boston, MA); J143 (anti-3), Dr.
L. J. Old (New York, NY); and E7P6 (anti-ß6), Dr. D.
Sheppard (San Francisco, CA). Monoclonal antibodies to 5
and v were purchased from Telios (San Diego, CA);
anti-vß3 and
anti-vß5 were purchased from Chemicon
(Temencula, CA); fluorescein-conjugated antimouse and antirabbit IgG
and horseradish peroxidase-conjugated antirabbit IgG were purchased
from Ortho (Raritan, NJ). Rabbit polyclonal antibodies to human FN,
collagen (CoG), vitronectin (VN), and laminin (LM) were purchased from
Chemicon.
Cells were plated onto sterile glass coverslips and cultured for up to 72 h at 37 C in RPMI-10% FCS. Cells were rinsed in PBS, fixed in 3.5% paraformaldehyde in PBS for 10 min, incubated in 0.5% Triton X-100 for 10 min, and blocked in 0.5% BSA for 10 min. Cells were incubated with phycoerythrin-conjugated phalloidin (Sigma) or primary antibody in PBS-0.2% Tween-20 for 1 h, washed in PBS, incubated with fluorescein-conjugated secondary antibody for 30 min, washed again, briefly rinsed in distilled water, mounted on microscope slides in PBS-50% glycerol, and observed with a fluorescence microscope (Zeiss, Oberkochen, Germany). Flow cytometric analysis was performed as follows. Cells harvested from subconfluent cell cultures by trypsin-PBS were incubated with the primary monoclonal antibody for 1 h at 4 C in 0.5% BSA-PBS, washed in the same buffer, and incubated again with the secondary fluorescein-conjugated antibody for 30 min at 4 C. Cells were resuspended in BSA-PBS and analyzed by flow cytometry. Nonspecific Igs of the same isotype were used as controls. The expression of each integrin was represented as the relative fluorescence index (RFI) = experimental mean fluorescence/control mean fluorescence.
Enzyme-linked immunoassay
Cells were plated in 96-well flat-bottomed microtiter plates (Costar, Cambridge, MA) in RPMI with or without serum, and after an appropriate time, the cells were fixed by methanol-acetone (vol/vol) for 10 min at room temperature and air-dried. Wells were filled with 100 µL 2% rabbit serum antihuman FN in PBS, 0.5% BSA, and 0.2% Tween-20 and allowed to react for 1 h at room temperature. Then, the plates were washed with PBS, filled with 100 µL horseradish peroxidase-conjugated antirabbit IgG in PBS and 0.2% Tween-20, allowed to react for 1 h, washed again with PBS, and filled with 150 µL 1 mg/mL o-phenylenediamine, 0.006% hydrogen peroxide, and 0.1 mol/L citrate buffer, pH 5.0. After 30-min incubation, the absorbance at 450 nm was measured by a spectrophotometer.
Cell attachment assay
The assay was performed in 96-well flat-bottomed microtiter plates. The wells were filled with 100 µL of the appropriate dilution in PBS of FN (Collaborative Research, Bedford, MA). After overnight incubation at 4 C, the plates were washed with PBS, filled with 100 µL 1% heat-denatured BSA, and incubated for 1 h at room temperature. Then, plates were washed and filled with 100 µL/well PBS, 0.9 mmol/L CaCl2, and 0.5 mmol/L MgCl2 containing 7 x 104 cells. After 30 min at 37 C, plates were gently washed three times with PBS, and the attached cells were fixed with 3% paraformaldehyde for 10 min, followed by 2% methanol for 10 min, and finally stained with 0.5% crystal violet in 20% methanol. After 10 min, the plates were washed with tap water, the stain was eluted with a solution of 0.1 mol/L sodium citrate, pH 4.2, in 50% ethanol, and the absorbance at 540 nm was measured by a spectrophotometer.
In the adhesion inhibition assay, 5 x 104 cells/well were coincubated with 100 or 500 µg/mL RGD-containing peptides (RGSP = Gly-Arg-Gly-Asp-Ser-Pro; RGTP = Gly-Arg-Gly-Asp-Thr-Pro) or RGE-containing peptides (Gly-Arg-Gly-Glu-Ser-Pro; Telios) in plates previously coated with 2 µg/mL FN. All experiments were performed in quadruplicate. Results were expressed as a percentage of the adhesion obtained in the absence of peptides.
DNA electrophoresis and estimation of apoptotic cells
Suspended cells collected by centrifugation and adherent cells were washed in PBS; lysed in 300 µL 0.5% Triton X-100, 5 mmol/L Tris buffer (pH 7.4), and 20 mmol/L ethylenediamine tetraacetate (TTE) for 20 min at 4 C; and centrifuged at 13,000 rpm for 30 min. Centrifugation-resistant low mol wt DNA was extracted with phenol-chloroform, precipitated with ethanol, and incubated with 0.5 µg/mL deoxyribonuclease-free ribonuclease for 30 min at 37 C. DNA with loading buffer was electrophoresed in 1% agarose and 1 µg/mL bromide at 50 V in 45 mmol/L Tris-borate and visualized by UV.
Cytofluorometric estimation of apoptosis was performed as described previously (21). Floating cells were collected, washed in cold PBS, added to adherent cells, and trypsinized. Cells were washed again in PBS and fixed in 70% cold ethanol for 30 min. Ethanol was removed by two PBS washes, and cells were incubated in PBS, 50 µg/mL propidium iodide, and deoxyribonuclease-free 10 µg/mL ribonuclease A overnight at 4 C. Cells were then analyzed by flow cytometry using a FACScan (Becton Dickinson Co.).
Statistics
Results are presented as the mean ± SD. SDs less than 10% are not reported in the diagrams.
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Results |
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The thyroid epithelial nature of the TAD-2 cell line was confirmed
by flow fluorocytometry using anticytokeratin and antithyroglobulin
antibodies. The presence of cytokeratin in the cells ascertained the
epithelial origin of the TAD-2 cell line (Fig. 1A
). Antithyroglobulin antibodies weakly
stained the cells, demonstrating, as expected, a low thyroglobulin
content (Fig. 1B), as TAD-2 cells originate from fetal thyroid, and TSH
was not present in the culture medium.
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The cell monolayer in in vitro culture is determined by
cell interaction with insoluble ECM components recruited from the serum
and/or produced by the cells themselves. To determine whether thyroid
cell anchorage was an integrin-RGD-dependent mechanism, 5 x
104 TAD-2 cells/well, obtained from subconfluent cultures
by mild trypsin treatment, were plated in 96-well flat-bottomed
microtiter plates in 100 µL RPMI-1% FCS. The cells were cultured in
the presence of synthetic peptides containing the sequence RGDSP that
inhibit integrin binding to both FN and VN, or in the presence
of control peptides containing the sequence RGESP or anti-ECM-purified
Igs (Fig. 2). After 8 h, nonadherent
cells were removed by gentle washing, adherent cells were observed by
inverted phase contrast microscope, and their number was determined as
described in Materials and Methods for the cell attachment
assays. Only a few round cells were present in the wells containing
RGDSP peptides or anti-FN antibody, whereas in the presence of
RGESP-containing peptides, the majority of the cells were adherent and
acquired a flattened shape. Cell adhesion was inhibited by RGDSP
peptides (92% inhibition), whereas RGESP peptides had no blocking
effect, demonstrating that cell anchorage to the plate was mediated by
a RGD-dependent FN/VN-integrin interaction. The anchorage
inhibition obtained by anti-FN antibodies was only slightly lower (88%
inhibition), suggesting that FN is the major ECM component involved in
TAD-2 cell anchorage, although a minor role for VN or other matrix
proteins cannot be excluded.
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Cells were plated in RPMI in the presence of 10% FCS on
coverslips and cultured for up to 72 h (Fig. 3). As expected, increasing the time of
culture resulted in a progressive flattening of cells and actin
microfilament organization. Cortical actin organized first, followed by
a progressively more complex cytoskeletal organization. A diffuse
staining was observed with anti-FN antibody after trypsin treatment in
uncultured cells (not shown). Trypsin treatment of cultures did not
completely remove the FN present on the cell surface, and intracellular
FN was also present in cultured cells 1 h after plating. Then,
increasing the time of culture resulted in progressive organization and
deposition of FN fibrils. After 72 h of culture, the cells were
wrapped in a dense net of FN fibrils deposited on both lower and upper
sides of the cells. The cells were also plated and cultured in medium
containing 0.2% BSA in the absence of FCS (not shown). In serum-free
cultures, the cells required several hours to became adherent and an
even longer time to spread and organize actin microfilaments. Cells
deposited FN fibrils, but their staining remained dim even after 3 days
of culture.
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A total of 3 x 104 cells were plated in
triplicate wells in RPMI medium-0.2% BSA with or without 1 mg/mL
soluble FN or with 1% FCS. At different times wells were washed, and
adherent cells and insoluble deposited matrix were fixed. By
enzyme-liked immunoassay with antihuman FN, the total amount of FN
present in the cells or deposited onto the bottom of the wells was
estimated (Fig. 4). After 1 h, FN
was already detectable in the FCS-containing wells, and increasing the
time of culture resulted in a progressive FN deposition. Serum is a
rich FN solution, and a 1% FCS solution contains about 3 µg/mL FN.
In serum-free medium containing human FN, FN was not deposited as
insoluble matrix, demonstrating that TAD-2 cells were not able to
recruit FN from the medium. In the absence of FCS, cells were not
adherent until 6 h and were removed by PBS washing. After that
time, a low amount of FN that remained constant during the culture was
detected.
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The expression of integrin-FN receptors was evaluated by flow
cytometry with monoclonal antibodies specific for the ß1,
ß6, 3, 4, 5,
v, vß3, and
vß5 chains. Figure 5 reports the average expression of
integrin heterodimers and integrin subunits measured in TAD-2 cells
cultured at subconfluence. 3 was the most abundant
integrin subunit (RFI = 218 ± 16.2); v was
strongly expressed, although at a lower level (RFI = 58.0 ±
2.1); whereas 5 was only slightly expressed (RFI =
6.1 ± 0.9), and 4 and ß6 were
totally absent. Monoclonal antibodies to whole integrin heterodimers
detected the presence of vß3, whereas the
VN receptor vß5 was totally absent. Cells
cultured for 3 and 72 h on coverslips in medium supplemented with
FCS were fixed and stained by indirect immunofluorescence with
antipaxillin, ß1, 3, 5,
v, and vß3 antibodies (Fig. 6). Paxillin, v, and
vß3 clearly localized in large focal
contacts, whereas anti-ß1 and anti-3
antibodies produced a fine dotted staining fairly distributed on the
entire cell surface. 5 was also localized in focal
contacts, but its staining was extremely dim compared with that of
v or paxillin (not shown).
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Cell attachment assays were performed in 96-well flat-bottom
microtiter plates coated with different concentrations of human FN
(Fig. 7A). Thyroid cells from
subconfluent cultures showed a concentration-dependent adhesion to FN,
reaching a maximum at about 12.5 µg/mL. The substrate concentration
required to achieve 50% of the maximal cell adhesion was about 2
µg/mL FN.
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Cell adhesion to FN was completely blocked in the presence of 100
µg/mL RGDSP or 500 µg/mL RGDTP peptides, whereas RGESP peptides had
no effect. Inhibition of integrin-mediated adhesion induces apoptosis
A total of 5 x 105 TAD-2 cells were plated in medium with 10% FCS, 10% FCS in a plate coated with a thin layer of 2% agarose to prevent matrix deposition, 0.2% BSA without FCS in a FN-coated plate (10 µg/mL in PBS overnight), or 50 µg/mL soluble FN-containing medium without serum. After a few hours in the presence of FCS and in the FN-coated plates, cells were adherent and acquired a flattened shape, whereas in the agar-coated plates and in the presence of soluble FN, the cells remained nonadherent, solitary cells showed a spherical conformation, and the majority clustered to form large cell aggregates floating in the medium.
After 24–96 h of culture, both floating and adherent cells were
collected, and apoptosis was estimated by flow cytometric analysis.
Hypodiploid cells were observed in the agar-coated plates and in the
presence of soluble FN, whereas they were not evident in the presence
of FCS or in the FN-coated plates. DNA fragmentation observed by DNA
electrophoresis confirmed apoptotic cell death (not shown). Apoptosis,
estimated by flow cytometric analysis, showed a time-dependent
increment that reached 95% after 72 h of culture in the
agar-coated plates in the presence of FCS as well as of soluble FN
(Fig. 8). Similar results were obtained
after 48 h of culture in serum-containing medium when adhesion to
FN was inhibited by RGD-containing peptides (Fig. 9). The cells were plated in RPMI medium
and FCS with or without 100 µg RGDSP, RGDTP, or RGE peptides, and
each 24 h, 100 µg more of the peptides were added to the
culture. Alternatively, the cells were cultured in the absence of
peptides in an agar-coated plate. In the presence of RGD peptides, as
in the agar-coated plates, the cells remained nonadherent and acquired
a spherical conformation or clustered to form floating aggregates.
Under these experimental conditions, inhibition of cell adhesion
induced apoptosis. RGE-containing peptides did not affect cell adhesion
and did not induce apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ECM deposition and basement membrane formation occur in cultures of many cell types. The ability of primary cultures of porcine thyroid cells to synthesize FN and type IV collagen has been described (23, 24). The differentiated rat thyroid epithelial cell line FRTL-5 synthesizes, secretes, and organizes an ECM containing FN, CoG, and also LM (25). Loss of or change in the synthesis and assembly of matrix proteins follows cell transformation, and aberrant distribution of ectopic matrix is a frequent finding in tumors, including thyroid tumors (22).
As shown in Fig. 1, adherence of TAD-2 cells in culture is mediated by
FN through one or more integrin receptors, although a minor role of
other matrix proteins cannot be excluded. TAD-2 cells, like primary
cultures of thyroid cells, can be cultured as a monolayer in the
presence of serum. Although serum contains a large amount of FN that
could be recruited by the cells, soluble FN was not used by TAD-2 to
adhere, as demonstrated in the experiment with soluble human FN.
Nevertheless, serum was required for active FN deposition. Trypsin
treatment of cultures did not completely remove the FN present on the
cell surface, as it could be detected by immunostaining after trypsin
treatment in uncultured cells, but although the total amount of FN was
very low and remained constant in serum-free cultures, it increased and
was actively deposited as insoluble matrix in the presence of serum.
This phenomenon was not restricted to thyroid cells and confirms
previous observations in mesenchymal cells. Serum stimulation of
quiescent fibroblasts induces coordinate transcriptional activity of
several cytoskeleton and extracellular matrix genes. ß-Actin,
-tropomyosin, and FN together with c-fos and
c-myc belong to the class of the so-called early growth
response genes whose expression is growth factor regulated (26, 27).
Also in AKR-2B cells, FCS, epidermal growth factor, insulin,
platelet-derived growth factor, transforming growth factor-ß, and
phorbol esters are mitogenic and strongly stimulate FN,
c-fos, and c-myc gene transcription within few
minutes, thus determining cell proliferation and increased synthesis of
FN (28).
As previously described in thyroid cells in primary cultures obtained
from thyroids of adult subjects (19), in TAD-2 cells,
3ß1 integrin was the most abundant FN
receptor. This integrin was distributed on the entire cell surface, but
did not localize in the focal contacts. This surface-diffused
distribution is characteristic of the 3ß1
integrin, and it has been described in a number of cell types, such as
fibroblasts and epithelial cells (29). Although several studies have
demonstrated a role of different FN receptors in regulating cell
proliferation and cell survival, none has demonstrated such functions
of the 3ß1 integrin; thus, this receptor
has to be considered to play only a structural role, whereas its
signaling capacity is not yet established (18, 30, 31, 32, 33, 34).
Although the 5 chain was only minimally expressed,
v was strongly represented. The
v integrin subunit associates with several
ß-subunits, including ß1 (35), ß3 (36),
ß5 (37), ß6 (38), and ß8
(39), generating monospecific receptors that bind only to FN or VN
(vß1 and vß5,
respectively) and receptors with multiple specificity interacting with
both substrates (vß3 and
vß6). The
vß1, vß3, and
vß6 receptors colocalize with paxillin,
p125FAK, and other molecules into adhesion plaques,
transmembrane structures linking the ECM with components of the
cytoskeleton (40). Although antibodies specific to
vß1 were not used in our study, the lack
of immunolocalization of the ß1-subunit into the focal
contacts suggests that the expression of
vß1 was very poor or totally absent in
TAD-2 cells. Among the other
v-subunit-containing integrins, only
vß3 was expressed, whereas
vß6, a FN receptor expressed predominantly
by epithelial cells (41) and vß5 (that
binds VN) were not found. The matrix interactions with
5ß1 and vß3
promote cellular responses such as proliferation and cell spreading
(36, 42, 43). In the present study we did not determine the specific
contribution of each integrin receptor; however, the experiments with
RGD-containing peptides supported the evidence that FN exerts its
biological role through integrins, because all these receptors interact
with their ligands in an RGD-dependent manner (34, 44). After binding
to their ligands, integrins cluster and promote the assembly of
cytoskeleton, actin microfilament polymerization, and cell spreading.
Endothelial and epithelial cells that are prevented from attaching to
an ECM substrate do not organize their cytoskeleton and undergo
apoptosis (45, 46). A number of studies report that proper cytoskeletal
organization and cell shape, and not the simple occupancy of integrin
receptors, are required to prevent apoptosis (45). Other studies
demonstrated that apoptosis could be prevented by the binding of
different types of integrins (vß3,
5ß1, and 2ß1)
to different substrates (FN, VN, and CoG), thus suggesting that this
phenomenon is not restricted to a specific integrin but, rather, is
generated by the ensuing cytoskeleton organization (47). Cell shape and
cytoskeletal organization are required for anchorage-dependent cell
survival, but this might not be sufficient, and specific intracellular
signaling could be required. Apoptosis of CHO cells is not prevented by
vß1 binding to FN, whereas
5ß1 binding to FN is effective in this
regard, thus demonstrating the existence of specific signaling (48).
This effect is independent of the level of p125FAK
phosphorylation, whereas it is associated with increased Bcl-2 protein
expression. The question of which integrin triggers signals that
prevent thyroid cells from entering into a suicide program has not been
addressed in the present study. As thyroid tumors display a changed
profile of integrin expression (49), the elucidation of this question
will be a valuable step toward a further understanding of the nature of
thyroid tumor malignancy.
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Acknowledgments |
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Footnotes |
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Received March 20, 1998.
Revised May 7, 1998.
Revised June 25, 1998.
Accepted July 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin and the ß1 integrin
subunit in vitro. J Cell Biol. 111:721–729.5ß1 integrin
receptor in the proliferative response of quiescent human melanoma
cells to fibronectin. Cancer Res. 52:4499–4506.2ß1 integrin expression results in
reduced cyst formation, failure of hepatocyte growth factor/scatter
factor-induced branching morphogenesis, and increased apoptosis. Cell
Sci. 108:3531–3540.[Abstract]
v subunit to form a novel vitronectin receptor in a
human embryonic kidney cell line. J Biol Chem. 265:5938–5941.vß6 integrin promotes
proliferation of colon carcinoma cells through a unique region of the
ß6 cytoplasmic domain. J Cell Biol. 127:547–556.5ß1 integrin supports survival of cells on
fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA. 92:6161–6165.This article has been cited by other articles:
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  M. Illario, A. L. Cavallo, S. Monaco, E. Di Vito, F. Mueller, L. A. Marzano, G. Troncone, G. Fenzi, G. Rossi, and M. Vitale Fibronectin-Induced Proliferation in Thyroid Cells Is Mediated by {alpha}v{beta}3 Integrin through Ras/Raf-1/MEK/ERK and Calcium/CaMKII Signals J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2865 - 2873. [Abstract] [Full Text] [PDF]
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 J.-B. Michel Anoikis in the Cardiovascular System: Known and Unknown Extracellular Mediators Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2146 - 2154. [Abstract] [Full Text] [PDF]
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 M. Illario, A. L. Cavallo, K. U. Bayer, T. Di Matola, G. Fenzi, G. Rossi, and M. Vitale Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation J. Biol. Chem., November 14, 2003; 278(46): 45101 - 45108. [Abstract] [Full Text] [PDF]
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T. Di Matola, F. D’Ascoli, G. Fenzi, G. Rossi, E. Martino, F. Bogazzi, and M. Vitale Amiodarone Induces Cytochrome c Release and Apoptosis through an Iodine-Independent Mechanism J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4323 - 4330. [Abstract] [Full Text]
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T. Di Matola, F. Mueller, G. Fenzi, G. Rossi, M. Bifulco, L. A. Marzano, and M. Vitale Serum Withdrawal-Induced Apoptosis in Thyroid Cells Is Caused by Loss of Fibronectin-Integrin Interaction J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1188 - 1193. [Abstract] [Full Text]
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 M. Vitale, T. Di Matola, F. D'Ascoli, S. Salzano, F. Bogazzi, G. Fenzi, E. Martino, and G. Rossi Iodide Excess Induces Apoptosis in Thyroid Cells through a p53-Independent Mechanism Involving Oxidative Stress Endocrinology, February 1, 2000; 141(2): 598 - 605. [Abstract] [Full Text] [PDF]
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M. Vitale, T. Di Matola, G. Rossi, C. Laezza, G. Fenzi, and M. Bifulco Prenyltransferase Inhibitors Induce Apoptosis in Proliferating Thyroid Cells through a p53-Independent, CrmA-Sensitive, and Caspase-3-Like Protease- Dependent Mechanism Endocrinology, February 1, 1999; 140(2): 698 - 704. [Abstract] [Full Text]
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