Integrin 
vß3 mediates K1735 murine melanoma cell motility in vivo and in vitro
Xiaowu Li1,
Joseph Regezi1,
F. Patrick Ross3,
Scott Blystone4,
Du
ko Ili
1,
Stanley P. L. Leong2 and
Daniel M. Ramos1,*
1
Department of Stomatology, University of California San Francisco, San
Francisco, CA 94143, USA
2
Department of Surgery, University of California San Francisco, San Francisco,
CA 94143, USA
3
Department of Pathology, Washington University School of Medicine, St Louis,
MO 63110, USA
4
Departments of Anatomy and Cell Biology, SUNY Health Science Center, Syracuse,
NY 13210, USA
*
Author for correspondence (e-mail:
dramos{at}itsa.ucsf.edu
)
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Fig. 1. K1735 primary tumor formation requires ß3 expression.
2x105 ß3-positive M2 cells or 2x105
ß3-antisense-expressing M2-Tß3 cells were injected transorally into
the floor of the mouth of syngeneic mice. After 4 weeks the mice were
sacrificed and evaluated for tumor formation. Note that the mice injected with
the M2 cells developed large primary tumors (A, left), whereas those injected
with the M2-Tß3 cells were considerably smaller (A, right). (B) The
average weight of tumors formed by M2 cells was 5.0 g (±1 g), whereas
tumors formed by the M2-Tß3 cells averaged 1.2 g (±0.4 g). 12 mice
were injected with each cell line.
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Fig. 2. K1735 melanoma metastasis requires expression of ß3 integrin.
2x105 M2 (A), M2-Tß3 (B), C23 (C), or C23-mß3 (D)
cells were injected subcutaneously into the hind flank of C3H/HeN mice. After
12 weeks the mice were sacrificed and analyzed for metastases. On average 100
colonies (±20) were formed by M2 (A, arrows) and 40 lesions
(±10) by the C23-mß3 cells (D, arrows). By contrast, M2-Tß3
(B) and C23 cells (C) did not form pulmonary metastases. Quantitation of the
experiment is shown in E.
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Fig. 3. Metastatic lesions formed by C23-mß3 cells retain ß3 integrin
expression. (A) In addition to pulmonary lesions, two mice injected with the
C23-mß3 cells developed cardiac metastases. H and E staining of the
lesions showed an infiltration by the tumor cells into the cardiac muscle. (B)
Sections of cardiac lesions were incubated with anti-ß3 antibodies
(without counterstaining) to localize the expression of this integrin. Note
the intense membrane staining (arrows) indicating continued expression of
ß3. The inset shows the area marked by the arrows at high
magnification.
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Fig. 4. ß3 integrin mediates autophosphorylation of FAK and the activity of
Src. The cells were plated on VN without serum for 30 minutes on all panels.
(A) Cells were extracted and immunoprecipitated with anti-FAK antibodies
followed by western blotting with anti-pTyr397 antibodies (top).
The membrane was stripped and reprobed with anti-FAK antibodies (bottom). (B)
The cell lysates were immunoprecipitated with anti-Src antibodies. The immune
complexes were analyzed for the incorporation of  -32P by
kinase assay (top). To ensure equivalent amounts of immunoprecipitated
protein, the lysates were immunoprecipitated followed by western blotting with
anti-Src antibodies (bottom). Autophosphorylation of Fyn was analyzed by
kinase assay (top). The relative amount of immunoprecipitated protein was
analyzed by an immunoprecipitation followed by western blotting with anti-Fyn
antibodies (bottom). (C) To detect putative complex formation, samples were
immunoprecipitated with anti-Src antibodies followed by western blotting with
antibodies to FAK (top). Relative amounts of immunoprecipitated protein were
determined by an immunoprecipitation followed by western blotting with
anti-Src antibodies (bottom). To detect FAK/Fyn complex formation, samples
were immunoprecipitated with anti-FAK antibodies followed by western blotting
with anti-Fyn antibodies (top). Relative amounts of Fyn were identified by
immunoprecipitation followed by western blotting with anti-Fyn antibodies
(bottom).
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Fig. 5. Expression of FRNK suppresses phosphorylation of FAK at Tyr397
and K1735 cell motility. M2 and M2GFP-FRNK cell lysates were
immunoprecipitated with anti-FAK and then analyzed by western blotting with
anti-pTyr397 antibodies (top). The membrane was stripped and
reprobed with anti-FAK antibodies to ensure that amounts of protein were
equivalent (middle). Detection of the FRNK construct was evaluated by a
western blot of whole cell lysate with antibodies to the C-terminus of FAK
(lane 2, bottom). (B) M2 and M2GFP-FRNK cells were seeded onto VN-coated
filters and monitored for migration over a 2 hour period or plated onto
RBM-coated filters and allowed to invade overnight, as described in Materials
and Methods. The results (means±s.e.m.) are representative of three
independent experiments each performed in triplicate. The average number of M2
cells migrating on VN was 238 (±11.5), whereas, the average number of
migrating M2GFP-FRNK cells was 11.5 (±1). Similarly, invasion of an RBM
was suppressed approximately 80% by expression of FRNK (B). The average number
of M2 cells invading was 46 (±19), whereas only 8 (±1)
M2GFP-FRNK cells invaded. These results suggest that phosphorylation of FAK on
Tyr397 is required for maximum K1735 cell motility.
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Fig. 6. K1735 cell migration requires active Src. (A) M2, M2KD-Src, C23, and
C23CA-Src cells were analyzed for autophosphorylation of Src by kinase assay
(top-left). Immunoprecipitation followed by western blotting using anti-Src
antibodies was done to detect changes in expression as a result of
transduction with the mutant constructs (bottom-left). Western blotting of
whole cell lysate was done to detect changes in Src expression as a result of
transduction with the Src constructs (top-right). Western blotting of whole
cell lysate with antibodies to paxillin was used as protein loading control
(bottom-right). (B) Migration of the four cell lines was evaluated on VN. The
results (means±s.e.m.) are representative of three independent
experiments each performed in triplicate.
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© The Company of Biologists Ltd 2001
