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Regular Articles |
From the Wistar Institute*
and the Department of
Pathology,
University of Pennsylvania,
Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 vitronectin receptor
on melanoma cells is associated with tumor thickness and the ability to
invade and metastasize. To address the role of vß3 in the complex
process of progression from the nontumorigenic radial to the
tumorigenic vertical growth phase of primary melanoma, we
examined the biological consequences of overexpressing vß3 in
early-stage melanoma cells using an adenoviral vector for gene
transfer. Overexpression of functional vß3 in radial growth phase
primary melanoma cells 1) promotes both anchorage-dependent and
-independent growth, 2) initiates invasive growth from the
epidermis into the dermis in three-dimensional skin
reconstructs, 3) prevents apoptosis of invading cells,
and 4) increases tumor growth in vivo. Thus,
vß3 serves diverse biological functions during the progression
from the nontumorigenic radial growth phase to the tumorigenic and
invasive vertical growth phase primary melanoma.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The successful isolation and in vitro propagation of cells derived from different stages of progression has provided an excellent experimental model for studying tumor progression.4-8 Cultured cells from RGP and VGP progression stages have biological properties that reflect the stage in vivo. Cells with RGP-like phenotype 1) require complex media containing several growth factors for continuous proliferation in culture due to their limited autoexpression of growth factors,7 2) do not grow in soft agar or form very few colonies,4,7 3) show a heterogeneous response to keratinocyte-mediated control of growth and regulation of cell surface receptor expression,9 and 4) are nontumorigenic or grow very slowly in immunodeficient nude or SCID mice to a small tumor mass (<150 µg) over 3 to 4 months.7 These results are consistent with analyses from patients indicating that RGP primary melanoma cells have a nonmalignant phenotype relatively similar to precursor cells.3 However, RGP primary melanoma cells are clearly distinguished from normal melanocytes or nevus cells by prolonged survival in culture or, as we have demonstrated with seven cell lines, by indefinite growth.7 Most, but not all, cell lines are also independent of basic fibroblast growth factor (bFGF) and the phorbol ester TPA for growth and survival.7
In contrast to cells from RGP lesions, those with a VGP-like phenotype are readily adapted to growth in tissue culture8 and need, at most, one growth factor (insulin-like growth factor (IGF)-1 or insulin) for proliferation.10 VGP cells express a variety of growth factors for either autocrine or paracrine stimulation11 and readily adapt to growth in growth-factor-free medium, leading to increased invasiveness through basement membranes in vitro and metastasis formation in vivo.12 VGP cells also form colonies in soft agar,4 do not respond to growth control by keratinocytes,9 and are tumorigenic in immunodeficient mice, with continuous local growth until the host dies.5,13
Integrins constitute a family of membrane glycoproteins that are
responsible for cell-extracellular matrix and cell-cell adhesion.
Accumulating evidence points to the role of integrins as signal
transducers in a variety of cellular events, including migration,
proliferation, survival, invasion, differentiation, and matrix
remodeling.14-18
Given their potential as diagnostic and
prognostic markers and as therapeutic targets, much effort has focused
on the differences in integrin profiles between normal and malignant
melanocytes.18-20
Among the most consistent observation is
the up-regulation of the ß3 subunit of the vß3 vitronectin
receptor in VGP melanoma in situ.21-24
In
addition, expression of vß3 correlates with clinical recurrence
and mortality.25,26
Immunohistochemical studies using
subunit-specific antibodies revealed that, in contrast to the selective
expression of ß3 subunit in advanced melanoma, the v subunit is
detected in all stages.21,23,26,27
The v subunit forms
complexes with ß1, ß3, ß5, and ß6, whereas ß3 in melanoma
predominantly pairs with v. Other experimental approaches, including
comparison of cell variants with different levels of vß3
expression23,28,29
and perturbation of vß3 function
with antibodies or peptides30-32
have further suggested
the contribution of vß3 to melanoma growth, invasion, and
metastasis. However, recent transfection studies aimed at dissecting
the biological role of the v and ß3 subunits in melanoma
progression have yielded conflicting results; transfection of v cDNA
into an v-deficient melanoma variant restored
tumorigenicity33
and promoted cell growth and survival in
three-dimensional collagen gel,34
whereas ectopic
expression of ß3 in a ß3-negative but highly metastatic human
melanoma cell line inhibited invasion and experimental
metastasis.35
To further investigate the potential role of vß3 in melanoma
progression, we overexpressed the ß3 subunit in RGP-like primary
melanoma cell lines using replication-deficient adenoviruses as a gene
delivery vehicle. We find that functional expression of the vß3
integrin receptor potentiates the malignant phenotype in
vitro and in vivo. In three-dimensional skin
reconstructs where the physiological melieu is recreated in
vitro, induced ß3 expression triggers an invasive phenotype and
prevents apoptosis. In vivo, ß3 overexpression induces
tumor growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human melanoma cell line WM1552C, which has an RGP-like phenotype,
was isolated as described3,28,29
from a superficial
spreading melanoma lesion. SBcl2, a primary melanoma cell line with an
RGP-like phenotype, was a gift from Dr. B. Giovanell (Stehlin
Foundation for Cancer Research, St. Joseph Hospital, Houston, TX).
WM1341D is a VGP-like cell line,8
which constitutively
expresses vß3. All cell lines were maintained in W489 medium
consisting of 4 parts MCDB153 supplemented with 2 mmol/L
CaCl2 and 1 part L-15, 5 µg/ml insulin, and 2% fetal
bovine serum (FBS). Keratinocytes were isolated from foreskin and grown
in serum-free keratinocyte growth medium (KGM) containing modified
MCDB15336
supplemented with bovine pituitary extract (BPE;
140 µg/ml), epidermal growth factor (EGF; 10 ng/ml), ethanolamine
(0.1 mmol/L), hydrocortisone (5 x 10-7 mol/L),
insulin (5 µg/ml), and O-phosphoryl ethanolamine (0.1 mmol/L).
Primary human dermal fibroblasts were initiated as explant cultures
from trypsin-treated and epidermis-stripped neonatal foreskin. These
cells were passaged in Dulbecco's modified Eagle's medium (DMEM) with
10% FBS. The trans-complementing 293 cells, a cell line
immortalized and transformed by adenovirus E1a and E1b, respectively,
were obtained from the Vector Core at the Institute for Human Gene
Therapy, University of Pennsylvania, Philadelphia, PA, and maintained
in DMEM with 10% FBS. All tissue culture reagents were purchased from
Sigma Chemical Co. (St. Louis, MO) except for EGF (Collaborative
Biomedical Products, Bedford, MA) and L-15 and DMEM (Gibco-BRL,
Gaithersburg, MD).
Construction of ß3/Ad5
Adenoviral vector ß3/Ad5 was constructed essentially as described.37 Briefly, a 4.0-kb EcoRI fragment containing the entire coding sequence of human integrin ß3 subunit in pBluescript was subcloned into EcoRI-cut pAd.CMV-Link.1 (obtained from the Vector Core, Institute for Human Gene Therapy). The resulting shuttle vector was linearized with NheI and co-transfected with ClaI-cut, E1–E3-deleted dl7001 human adenoviral DNA into 293 cells by standard calcium phosphate precipitation. Two days after transfection, cells were overlaid with 0.8% agar in MEM (Gibco-BRL) and fed every 3 to 4 days. Individual plaques were picked and screened for ß3 expression by immunoprecipitation with monoclonal antibody (MAb) SSA6 (kindly provided by Dr. J. Hoxie, University of Pennsylvania).38 Positive clones were subjected to three rounds of plaque purification to eliminate contamination with wild-type virus. Plaque-purified viruses propagated in 293 cells (ß3/Ad5) were purified by ultracentrifugation in a cesium chloride gradient as described.37 Viral titer was evaluated by absorbance at 260 nm, and the activity was assessed by plaque formation in permissive 293 cells.
Radiolabeling and Immunoprecipitation
Cells were infected at a multiplicity of infection of 10 and metabolically labeled by overnight incubation in methionine-free DMEM supplemented with 25 µCi/ml [35S]methionine (Amersham, Arlington Heights, IL). Cells were washed with PBS and extracted with non-ionic detergent buffer (10 mmol/L Tris/acetate, pH 8.0, 150 mmol/L NaCl, 0.5% Nonidet P-40, 0.5 mmol/L CaCl2) containing a protease inhibitor (2 mmol/L phenylmethylsulfonyl fluoride). Cell extracts were clarified by centrifugation at 14,000 x g for 20 minutes and precleared with protein-A-conjugated Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) for 30 minutes at 4°C. Precleared cell extracts were normalized according to radioactivity, and 100 µl was incubated with 1 µg of ß3-specific SSA6 MAb for 1 hour at 4°C. Protein-A-conjugated Sepharose beads were then added to the immune complexes. The mixture was incubated for an additional hour at 4°C followed by washing five times with DOC wash (50 mmol/L Tris/HCl, pH 7.5, 150 mmol/L 1% Triton X-100, 5% deoxycholate, and 0.1% SDS). Antigens were released from the beads by boiling in Laemmli sample buffer. Samples were separated on 6% polyacrylamide gels under nonreduced conditions. Gels were dried and exposed to x-ray film.
Flow Cytometry
Infected cells were trypsinized, washed, and resuspended in serum-free DMEM with 10 µg/ml MAb SSA6. After 1 hour of incubation at 4°C with gentle rocking, cells were washed to remove unbound antibodies and stained with 10 µg/ml fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at 4°C. After washing, cells were resuspended in PBS and analyzed by fluorescence-activated cell sorting (FACS) using an Ortho Cytofluorograf 50H connected to a 2150 Data Handling System (Ortho Diagnostics, Westwood, MA).
Anchorage-Dependent Growth Assay
Cells from subconfluent cultures were trypsinized and seeded in triplicate 35-mm wells at 2 x 105 cells/well. The medium was changed twice a week. At different time points, cells were harvested and counted in a Coulter counter (Coulter Electronics, Luton, UK). All assays were performed in triplicate wells.
Soft Agar Growth
To prevent cell attachment, 1 ml of 0.5% Agar Nobel (Difco Laboratories, Detroit, MI) in WM489 medium supplemented with 50 µg/ml BPE, 3.5 ng/ml EGF, and 7% FBS was placed in six-well tissue culture plates and allowed to gel at room temperature. Subconfluent cultures were harvested by trypsinization, resuspended to 30,000 cells/ml in W489 medium supplemented with 5 ng/ml EGF and 70 µg/ml BPE, and mixed with agar to a final concentration of 6000 cells/well in 0.25% agar. Triplicate wells were prepared for each group of transduced and nontransduced cells.
Invasion and Cell Survival in Skin Reconstructs
Skin reconstructs were prepared as described39-41 with modifications. Briefly, subconfluent dermal fibroblasts isolated from foreskins were harvested and resuspended to 1.5 x 105 cells/ml in DMEM with 10% FBS and 1 mg/ml neutralized rat tail collagen (Collaborative Biomedical Products). Three milliliters of the mixture was then seeded onto Transwell inserts (Corning Costar Corp., Cambridge, MA) placed in six-well tissue culture plates with 1 ml of precast acellular collagen and incubated at 37°C. After 6 days, fibroblasts had contracted collagen gels, creating a concave surface that served as a cradle for seeding epidermal cells. This portion represented the dermal reconstruct. The expelled medium was suctioned off, and the dermal reconstruct was equilibrated in epidermal growth medium (EGM) composed of 3 parts DMEM, 1 part Ham's F-12, and 0.3% dialyzed newborn calf serum supplemented with 10 ng/ml EGF, 1.88 mmol/L CaCl2, 0.18 mmol/L adenine, 4 mmol/L glutamine, 53 nmol/L selenic acid, 0.1 mmol/L ethanolamine, 0.1 mmol/L O-phosphoryl ethanolamine, 5 µg/ml insulin, 5 µg/ml transferrin, 20 pmol/L tri-iodothyronine, 0.4 µg/ml hydrocortisone, 10 nmol/L progesterone, and 1.5 mmol/L HEPES for 1 hour at 37°C. The medium was discarded, and dermal reconstructs were dried at room temperature for 30 minutes. Melanoma cells were trypsinized and washed in Ca2+/Mg2+-free HEPES-buffered saline solution three times before mixing with keratinocytes at a 1:5 ratio in EGM to yield a cell concentration of 3 x 106/ml, and 50 µl of cell suspension was seeded onto the dry surface of dermal constructs. After 2 hours, cultures were submerged and re-fed every 2 to 3 days thereafter. Five days after seeding, medium was switched to maintenance medium (1:1 mixture of DMEM and Ham's F12 supplemented with 1% newborn calf serum, 1.95 mmol/L CaCl2, 0.18 mmol/L adenine, 4 mmol/L glutamine, 53 nmol/L selenic acid, 0.1 mmol/L ethanolamine, 0.1 mmol/L O-phosphoryl ethanolamine, 5 µg/ml insulin, 5 µg/ml transferrin, 20 pmol/L triiodothyronine, 0.4 µg/ml hydrocortisone, and 1.5 mmol/L HEPES), and cultures were lifted to the air-liquid level to allow further epidermal stratification for another 10 days with regular feeding. Skin reconstructs were then harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. The invasive capacity of melanoma cells was determined by morphological evaluation using hematoxylin and eosin staining. Cell survival after invasion into dermal reconstructs was assessed using an ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD).
Tumorigenicity
Melanoma cells were suspended at 3 x 108/ml in growth medium. Female SCID mice (five mice per group) were injected subcutaneously in the back with 100 µl of cell suspension. Tumor volume was determined as follows: (maximal dimensions x minimal dimensions)2/2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 Integrin by Melanoma Cells
Adenoviral vector ß3/Ad5 induced cell surface expression of ß3
within 48 hours of cell infection at a multiplicity of infection of 10
to 20, as evidenced by the increase in positively stained cells from
8.9% to 67.8% and 0.9% to 90.4% in WM1552C and SBcl2 cells,
respectively, in FACS analysis using lacZ/Ad5-infected cells
as controls (Figure 1A)
. Little
overexpression was found in WM1341D cells, which constitutively express
vß3. Immunoprecipitation analysis using a ß3-specific MAb and
extracts of radiolabeled cells normalized according to radioactivity
revealed bands with molecular masses corresponding to v and ß3 in
all samples (Figure 1B)
. Consistent with the FACS data, ß3/Ad5
induced a marked increase in vß3 expression in WM1552C (lanes 3
and 4) and SBcl2 (lanes 1 and 2) cells as compared with their control
virus-infected counterparts. Again, further up-regulation of ß3 in
vß3-expressing WM1341D cells by ß3/Ad5 was limited (lanes 5 and
6). In addition, immunoblotting of ß3 precipitates using an
v-specific MAb (a kind gift of Dr. S. L. Goodman, E. Merck,
Darmstadt, Germany) confirmed a functional association between
transduced ß3 and endogenous v in ß3/Ad5-infected SBcl2 cells
(data not shown).
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To investigate the role of the ß3 integrin subunit in growth
regulation of melanoma cells in vitro, cell proliferation in
monolayer culture and in soft agar was examined. Growth of monolayer
cultures was stimulated (twofold) in ß3-transduced WM1552C cells but
not in SBcl2 cells (Figure 2)
, whereas in
soft agar assays, colony-forming efficiencies were enhanced in both
cell lines, with a 3.5- and 1.5-fold increase in WM1552C and SBcl2 cell
growth, respectively (Figure 3)
.
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As integrin function is profoundly influenced by the extracellular
milieu,42-49
we examined melanoma invasion under
physiological conditions by incorporating the transduced cells into
three-dimensional skin reconstructs. Skin reconstructs consist of
artificial skin rebuilt from isolated cell populations and composed of
a stratified, terminally differentiated epidermal compartment of
keratinocytes and melanocytes, a dermal compartment consisting of
fibroblasts embedded in collagen gel, and a well established basement
membrane deposited by skin cells.41
ß3/Ad5-infected SBcl2
cells invaded deep into the dermis and formed cell nests (Figure 4, A and C)
, whereas
lacZ/Ad5-infected cells spread only horizontally (Figure 4, B and D)
. Moreover, lacZ/Ad5-infected cells showed the clear
morphological signs of apoptosis, including nuclear condensation,
membrane blebbing, and apoptotic bodies (Figure 4D)
. These cells
exhibited intense ApopTag staining, confirming their apoptotic cell
death (Figure 4F)
, whereas ß3-expressing SBcl2 cells were completely
negative (Figure 4E)
.
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To study the biological consequences of ß3 overexpression in
early-stage, nontumorigenic melanoma in vivo, tumorigenicity
of virus-transduced cells was evaluated in SCID mice injected
subcutaneously with 3 x 107
ß3/Ad5- or
lacZ/Ad5-infected cells. The average size of tumors formed
by ß3-expressing cells at 7 days after injection was 5-fold (WM1552C)
and 15-fold (SBcl2) larger than their lacZ/Ad5-infected
counterparts (Figure 5)
. However, due to
the episomal nature of adenoviral vectors, all tumors began
disappearing after day 10.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v that potentiates the malignant phenotype reminiscent of
the progression from RGP to VGP; ie, ß3-expressing cells exhibit a
growth advantage in monolayer cultures and soft agar, increased
invasiveness and survival in skin reconstructs in vitro, and
enhanced tumorigenicity in vivo.
Previous transfection studies showed that introduction of either
subunit of the vß3 receptor into metastatic melanoma cell lines
did not affect cell proliferation in vitro.33,35
In contrast, we find that overexpression of ß3 stimulates in
vitro growth of WM1552C cells. This is not surprising as interplay
between vß3 and growth factor receptors such as insulin and IGF
receptors50
as well as platelet-derived growth factor
(PDGF)-ß receptors51
has been shown to control cell
growth. However, the same growth-promoting effect was not seen in SBcl2
cells, which have a more rapid basal proliferation rate than that of
WM1552C cells (Figure 2)
. Thus, mechanisms other than vß3 appear
to be involved in SBcl2 cell growth stimulation. Indeed, given the
apparent redundancy of integrins, it is conceivable that functions of
vß3 are replaced in part by 5ß1,46
vß1,
vß5, or vß6,52
which are all present in melanoma
cells.
A hallmark of malignant transformation is anchorage-independent growth
of the cells. By introducing the ß3 integrin subunit, we demonstrated
an increase in colony-forming efficiency of early-stage melanoma cells
in soft agar. The mechanism(s) of this growth advantage after ß3
transduction are not clear. In melanoma, cell-cell interactions occur
through the adhesion receptor Mel-CAM, a member of the Ig gene
superfamily, which binds to an unknown ligand also expressed by
melanoma cells.9,53
vß3 expressed by melanoma cells
can bind to L1, another member of the Ig gene superfamily found on
melanoma cells.34
It is possible that such cell-cell
interactions may provide signals for anchorage-independent survival and
growth.
Progressive invasion into the dermis is one of the most important
characteristics of VGP melanoma cells. This process requires
disassociation of melanoma cells from neighboring keratinocytes and
attachment to and proteolytic degradation of basement membrane
components, followed by invasion and proliferation in the dermis. In
our skin reconstruct model, which, unlike traditional invasion assays,
accounts not only for tumor-cell-derived mechanisms but also for
microenvironmental factors from stromal cells, control virus-infected
SBcl2 cells grew in a pattern resembling RGP lesions, whereas
ß3-expressing cells showed a VGP growth pattern. The latter cells
invaded and proliferated deep in the dermis without signs of apoptotic
change whereas control cells remained in the epidermis, dying by
apoptosis in the dermis. Coordinate expression and activation of
vß3 with that of matrix metalloproteinases and urokinase-type
plasminogen activator receptor46,47
may play a crucial role
in proteolysis of the extracellular matrix during invasion. Indeed,
activated matrix metalloproteinase (MMP)-2 binds to cell surface
vß3, thereby localizing its enzymatic activity to the leading edge
of tumor cells.54
Montgomery et al34
have
provided evidence that survival and proliferation of
vß3-expressing melanoma cells in a three-dimensional collagen
matrix is mediated through the ligation of the collagen proteolytic
products to the cryptic binding site of vß3. Taken together, these
findings suggest a role for the vß3 integrin in melanoma
progression toward an increasingly aggressive phenotype.
In our study, tumorigenicity of early-stage melanoma cells was
increased after overexpression of the ß3 subunit. This finding
contrasts with previous transfection studies that identified v as
the crucial component in conferring an aggressive
phenotype33
or that report decreased metastatic potential
on ß3 transduction.35
The discrepancies might reflect a
variability in the degree and aspect of phenotype modulation
with the stage of progression, despite the profound effect of
vß3 expression on the biological properties of melanoma cells.
Indeed, metastatic cells are less susceptible than nontumorigenic RGP
primary melanoma cells to alterations induced by vß3
overexpression. Thus, the biological functions of vß3 in melanoma
may depend, in part, on the cellular background of a given stage of
tumor progression.
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Footnotes |
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Supported by NIH National Cancer Institute grants CA-47159 and CA-10815.
D.-T. Shih's current address: Department of Biology, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877.
J.-Y. Hsu's current address: Sidney Kimmel Cancer Center, San Diego, CA 92121.
Accepted for publication August 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 fibronectin- and vß3 vitronectin-receptor expression in melanocytic tumour progression. Histopathology 1994, 24:249-256[Medline]
v-integrins in human melanoma: gain of vß3 and loss of vß5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice [published erratum appears in Int J Cancer 1995, 62:365]. Int J Cancer 1995, 61:491-496[Medline]
vß3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions. Cancer Res 1997, 57:1554-1560vß3 integrin in human melanoma cell invasion. Proc Natl Acad Sci USA 1992, 89:1557-1561v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice. J Cell Sci 1995, 108:2825-2838[Abstract]
v gene expression in human melanoma tumorigenicity. J Clin Invest 1992, 89:2018-2022
vß3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen. Proc Natl Acad Sci USA 1994, 91:8856-8860vß3-negative human melanoma cell line inhibits invasion and experimental metastasis. Biochem Biophys Res Commun 1996, 226:75-81[Medline]
vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994, 79:1157-1164[Medline]
vß3 and 5ß1 integrins during human melanoma cell invasion. Cancer Res 1993, 53:3411-3415vß3. Cell 1996, 85:683-693[Medline]
vß3 integrin associates with activated insulin and PDGFß receptors and potentiates the biological activity of PDGF. EMBO J 1997, 16:5600-5607[Medline]
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R. A. Sturm, K. Satyamoorthy, F. Meier, B. B. Gardiner, D. J. Smit, B. Vaidya, and M. Herlyn Osteonectin/SPARC Induction by Ectopic {beta}3 Integrin in Human Radial Growth Phase Primary Melanoma Cells Cancer Res., January 1, 2002; 62(1): 226 - 232. [Abstract] [Full Text] [PDF]
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C. Berking, R. Takemoto, H. Schaider, L. Showe, K. Satyamoorthy, P. Robbins, and M. Herlyn Transforming Growth Factor-{beta}1 Increases Survival of Human Melanoma through Stroma Remodeling Cancer Res., November 1, 2001; 61(22): 8306 - 8316. [Abstract] [Full Text] [PDF]
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J. Brummer, A. Ebrahimnejad, R. Flayeh, U. Schumacher, T. Loning, A.-M. Bamberger, and C. Wagener cis Interaction of the Cell Adhesion Molecule CEACAM1 with Integrin {beta}3 Am. J. Pathol., August 1, 2001; 159(2): 537 - 546. [Abstract] [Full Text]
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S. Silletti, F. Mei, D. Sheppard, and A. M.P. Montgomery Plasmin-sensitive Dibasic Sequences in the Third Fibronectin-like Domain of L1-Cell Adhesion Molecule (CAM) Facilitate Homomultimerization and Concomitant Integrin Recruitment J. Cell Biol., June 26, 2000; 149(7): 1485 - 1502. [Abstract] [Full Text] [PDF]
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M.-Y. Hsu, F. E. Meier, M. Nesbit, J.-Y. Hsu, P. Van Belle, D. E. Elder, and M. Herlyn E-Cadherin Expression in Melanoma Cells Restores Keratinocyte-Mediated Growth Control and Down-Regulates Expression of Invasion-Related Adhesion Receptors Am. J. Pathol., May 1, 2000; 156(5): 1515 - 1525. [Abstract] [Full Text] [PDF]
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L. C. L. T. van Kempen, J. J. van den Oord, G. N. P. van Muijen, U. H. Weidle, H. P. J. Bloemers, and G. W. M. Swart Activated Leukocyte Cell Adhesion Molecule/CD166, a Marker of Tumor Progression in Primary Malignant Melanoma of the Skin Am. J. Pathol., March 1, 2000; 156(3): 769 - 774 |
