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Cell Growth & Differentiation Vol. 12, 99-107, February 2001
© 2001 American Association for Cancer Research

Integrin Expression and Usage by Prostate Cancer Cell Lines on Laminin Substrata1

Magnus Edlund2, Tadayuki Miyamoto2, Robert A. Sikes, Roy Ogle, Gordon W. Laurie, Mary C. Farach-Carson, Carol A. Otey, Haiyen E. Zhau and Leland W. K. Chung3

Departments of Urology [M. E., T. M., R. A. S., H. E. Z., L. W. K. C.] and Cell Biology [R. O., G. W. L., L. W. K. C.], University of Virginia Health System, Charlottesville, Virginia 22908; Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545 [C. A. O.]; and Department of Biological Sciences, University of Delaware, Newark, Delaware 19716 [M. C. F-C.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
During prostate cancer progression, invasive glandular epithelial cells move out of the ductal-acinar architecture and through the surrounding basement membrane. Extracellular matrix proteins and associated soluble factors in the basal lamina and underlying stroma are known to be important regulators of prostate cell behaviors in both normal and malignant tissues. In this study, we assessed cell interactions with extracellular matrix and stromal factors during disease progression by characterizing integrin usage and expression in a series of parental and lineage-derived LNCaP human prostate cancer cell lines. Although few shifts in integrin expression were found to accompany disease progression, integrin heterodimer usage did change significantly. The more metastatic sublines were distinct in their use of {alpha}vß3 and, when compared with parental LNCaP cells, showed a shift in {alpha}6 heterodimerization, a subunit critical not only for interaction with prostate basal lamina but also for interaction with the bone matrix, a favored site of prostate cancer metastases.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cancerous prostate cells are regulated in their differentiation, growth, and metastasis by interactions with the surrounding cells and ECMs4 (1, 2, 3) . Cell behavior decisions, such as decreasing cell-cell and cell-substrate attachment, and increasing cell motility are accompanied by changes in the expression and/or usage of adhesion receptors, including those of the integrin family (2 , 3) . Past studies in prostate cancer have focused on quantitation as well as cell surface distribution of integrins (4, 5, 6, 7, 8) and on correlating changes in integrin expression with invasive cell behavior (9 , 10) . The expression level studies have taken two forms: (a) cell lines with different metastatic potential have been found to express different levels and subtypes of integrins; and (b) within a given cell line, metastatic potential has been experimentally correlated with increases or decreases in levels of integrin expression (9 , 11 , 12) .

Integrin molecular structure, heterodimerization, and intra- and extracellular interactions of integrins with cytoplasmic regulatory proteins and ECM ligands provide tremendous potential for variation among cell types, well beyond that available through quantitative variation in integrin expression level alone. Integrins are themselves heterodimeric molecules, consisting of one {alpha} and one ß subunit, with at least 20 different combinations already described, many of which differ in their extra- and intracellular binding specificities (13 , 14) . "Inside-out" regulation of integrin heterodimer activity and subunit partner choices are thought to depend on unique cytoplasmic regulatory protein repertoires, which differ among host cell types (Refs. 15, 16, 17 and Ref. 18 and references within). "Outside-in" regulation by integrins, in response to extracellular cues, has also been well studied and has revealed shifts in integrin gene expression as well as changing integrin associations with numerous signaling molecules, including protein tyrosine kinases (focal adhesion kinase and pp60src), serine kinases (protein kinase C, extracellular signal-regulated kinase, c-Jun-NH2-terminal kinase, and integrin-linked kinase), and lipid intermediates (phosphatidylinositol 3'-kinase and phosphatidylinositol 4,5-kinase; Refs. 14 and 19, 20, 21 and the references within). Hence, integrin activity within a given cell is tightly coordinated with its cell cycle, gene expression profiles, differentiation, and cell survival (13) . The stroma is a source of key extracellular cues (including soluble growth factors and insoluble matrix proteins) known to modulate integrin-dependent cell functions (22 , 23) . Although a number of integrin variations during prostate cancer cell progression have been described (5, 6, 7, 8, 9, 10, 11, 12) , neither modulation of these variations by external factors nor integrin heterodimer usage regulation is well understood.

The LNCaP lineage cell model of prostate cancer progression (24, 25, 26) has given us an opportunity to follow coordinated changes in integrin expression, usage, and cell behavior of prostate cancer cells when exposed to different ECM substrata and stromally secreted soluble factors. LNCaP and LNCaP-derived cell lines are unique in that they vary in metastatic potential but share a common genetic background. Previous phenotypic (26) and genotypic (27) characterizations of these cell lines have revealed their remarkable resemblance to progressing clinical human prostate cancer. We focus here on characterizing interactions between these cancerous prostate cells and their ECM microenvironments, particularly on the ability of cell lines of different metastatic potential to attach, spread, and migrate on laminin, a key protein in both the basement membrane surrounding the acini and in tumors themselves (28) . We also examine cell line behaviors on several other matrix components found in bone, a favorite destination for prostate cancer cells following a metastatic cascade (Ref. 29 and the references within). Human prostate tumors disseminated to the bone have been shown to have altered integrin expression, particularly laminin-binding integrin expression, when compared with hyperplastic, benign tumors (11 , 12 , 30 , 31) . One integrin heterodimer thought to bind laminin along with VN is {alpha}vß3, an integrin that is not expressed in normal prostate tissue but is up-regulated in prostatic adenocarcinoma (11 , 32) . Likewise, in primary prostate carcinomas, shifts in {alpha}6 integrin subunit expression (and heterodimerization with its ß subunit partner) were observed during prostate cancer progression (8) . In other tumor cell types, the laminin-binding integrins {alpha}6ß4 and {alpha}6ß1 have also been linked to acquisition of invasive behaviors (6 , 12) .


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
LNCaP Parental and Lineaged Cell Lines Attach to Laminin Using Different Integrin Subunits.
Nonmetastatic LNCaP human prostate cancer epithelial cells and their derivative metastatic sublines (C4, C4-2, and C4-2B) readily attached to a common laminin substrate, and all displayed focal contacts and some poorly developed stress fibers, as seen by staining for filamentous actin. The poor development of stress fibers is characteristic of all LNCaP lineage-related cell lines and is not substrate dependent. Representative actin staining in attached LNCaP and C4-2 cells is shown in Fig. 1Citation ; C4 and C4-2B cells stained similarly (data not shown).



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Fig. 1. Actin staining in LNCaP and C4-2 cell lines. Both LNCaP and C4-2 cells show diffuse actin staining and small actin fibers when grown on laminin substrata labeled with phalloidin.

 
To identify the integrins used for attachment by the different cell types, parental LNCaP and its derivative C4, C4-2, and C4-2B cell lines were selected (26) , and specific, function-blocking integrin antibodies were added to the attachment assays. Although the antibody staining suggested the formation of focal adhesion structures in all cell lines (data not shown), the cells responded differently to the function-blocking antibodies (Fig. 2)Citation . Attachment of parental LNCaP cells was best blocked by antibodies against subunits {alpha}6 and ß4, whereas antibodies against these subunits did not effectively block attachment of C4, C4-2, or C4-2B cells, whose attachments were best blocked by antibodies against the intact {alpha}vß3 integrin and the subunits {alpha}3 and ß1. Attachments of all four cell lines were also somewhat reduced by antibodies against the subunit {alpha}2.



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Fig. 2. Inhibition of cell attachment to a laminin substrate using integrin subunit-specific antibodies. LNCaP and the derived sublines C4, C4-2, and C4-2B were preincubated with function-blocking antibodies as indicated. Experimental attachment is shown as a percentage of control, untreated cell attachment, and results are presented as the mean of representative triplicate experiments, with SDs shown as error bars.

 
The Differences in LNCaP and C4-2 Cell Attachment Are Not Likely to Be Due to Differential Expression of Integrin Subunits.
FACS analyses were used to determine integrin subunit ({alpha}2, {alpha}3, {alpha}v, ß1, ß3, and ß4) expression levels in LNCaP and C4-2 cell lines (Table 1)Citation . Characterization of potential laminin-binding integrin levels by flow cytometry revealed only one difference in expression level (i.e., the {alpha}2 subunit) among the four cell types. Although the expression of the integrin {alpha}2 subunit in C4-2 cells was approximately double that in LNCaP cells, all other integrin receptors were found to remain fairly constant in expression level across all cell lines (Table 1Citation ; including C4 and C4-2B; data not shown).


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Table 1 Expression of integrins by FACS analysis

Integrin expression in LNCaP and its more metastatic, derived C4-2 cell line. Values for integrin expression are presented as the mean of two individual duplicate experiments, with the range given in parentheses. An isotype nonspecific antibody was used for control. All experimental fluorescence values are reported as the ratio of the control and specific fluorescence values. Relative values of integrin expression can only be compared for the same antibody on different cells due to differences of antibody affinities for their ligands.

 
Cell surface expression data were verified by immunoprecipitation of integrin subunits {alpha}3, {alpha}6, {alpha}vß3, and ß1 from biotinylated cells of different cell lines (Fig. 3)Citation . Similar levels of the {alpha}3 and ß1 subunit were precipitated in all cell lines. The {alpha}3 subunit dimerizes most readily with the ß1 subunit, as seen by immunoprecipitation with either {alpha}3-specific or ß1-specific antibodies (Fig. 3A)Citation . Although immunoprecipitation with an {alpha}6 antibody coprecipitated ß1 and ß4 subunits from both LNCaP and C4 cells (Fig. 3B)Citation , the {alpha}6ß1 heterodimer is not likely to be used for laminin attachment in LNCaP cells because very little inhibition of cell attachment is seen by the ß1 antibody in LNCaP competition experiments (Fig. 2)Citation . In comparison with LNCaP, very little ß4 subunit appears to be used for laminin attachment in the C4-2 and C4-2B sublines; the {alpha}6 antibody did not immunoprecipitate as much of the ß4 subunit from the latter two cell lines (Fig. 3B)Citation , and a function-blocking antibody against ß4 did not inhibit their attachment to a laminin substrate (Fig. 2)Citation , as it does for LNCaP. Ratio comparisons, using band intensities on Western blots of {alpha}6-immunoprecipitated ß1 and ß4, reveal a 1:1 ratio of ß14 in LNCaP cells but show a ratio of 1:0.8 in C4 and 1:0.2 in C4-2 and C4-2B cells.



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Fig. 3. Immunoprecipitation of biotinylated cell surface integrin isotypes using {alpha}3 and ß1 (A), {alpha}6 (B), and {alpha}vß3 (C). Retrieved complexes from each LNCaP, C4, C4-2, or C4-2B cell line were separated by PAGE under reducing conditions, blotted, and visualized with peroxidase-conjugated streptavidin.

 
Although FACS analyses detected both {alpha}v and ß3 subunits in all cell lines, at equivalent surface expression levels, immunoprecipitation with antibody to the {alpha}vß3 heterodimer revealed nearly undetectable levels of {alpha}vß3 in LNCaP cells while readily detecting the heterodimer in all three derived sublines (Fig. 3C)Citation . Use of the {alpha}vß3 heterodimer does appear to be important for laminin attachment in the three metastatic sublines (but not LNCaP cells) because function-blocking antibody was able to inhibit cell attachment in the sublines (Fig. 2)Citation .

The {alpha}vß3 Subunit Is Necessary for C4-2, but not LNCaP, Cell Attachment and Migration.
Because prostate cancer cells metastasize preferentially to bone, we were particularly interested in the integrin heterodimers known to interact with VN and OPN, two noncollagenous bone matrix proteins. The integrin {alpha}vß3 was chosen for attachment and migration assays because it is known to interact not only with these two bone matrix proteins but also with laminin (33) . LNCaP and C4-2 cells adhered to all three substrata, but only C4-2 attachment could be inhibited with increasing concentrations of antibodies against {alpha}vß3 integrin (Fig. 4)Citation . At high antibody concentrations of 10 µg/ml, attachments of the metastatic C4-2 cells to all three substrata were reduced by approximately 60%, but no attachment effect was seen for the nonmetastatic LNCaP cells. However, LNCaP attachment could be decreased by using a {alpha}vß5 function-blocking antibody (data not shown). The role of the {alpha}vß3 heterodimer in cell migration was evaluated using modified Boyden chambers, and the haptotactic responses of each cell line were quantified on laminin, VN, and OPN. Fig. 5Citation shows the cell migratory behaviors of C4-2 cells on these three bone matrix proteins and that C4-2 cell migration could be inhibited by an {alpha}vß3 isotype-specific integrin antibody. LNCaP cells migrated at very low levels on both laminin and VN but did not migrate at all on OPN (data not shown).



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Fig. 4. Antibody-mediated attachment inhibition of LNCaP and C4-2 cells on VN or OPN substrata. Inhibition of attachment is shown with increasing {alpha}vß3 antibody concentration expressed as a percentage of control, untreated cell attachment. Values are the mean of two experiments (n = 6), and error bars represent SDs. Statistically significant differences from the control were at P < 0.001 (*).

 


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Fig. 5. Migration of C4-2 cells on laminin, VN, or OPN substrata in the presence and absence of 10 µg/ml control or {alpha}vß3 function-blocking integrin antibody. Boyden chambers were used for haptotactic assays, and values shown are the average of two experiments (n = 6). Error bars represent SDs. Statistically significant differences between {alpha}vß3 and the control were at P < 0.001 (*).

 
Soluble Stromal Factors Induce C4-2, but not LNCaP, Cells to Attach to Laminin.
To begin identifying possible regulators of integrin subunit usage and cell behavior in LNCaP and C4-2 cell lines, we tested the effect of stromal factors on cell line interactions with laminin substrata. Cells were treated with conditioned media from primary cultures of the transition or peripheral zone stromal cells of the prostate gland from four different patients with prostatic adenocarcinoma and allowed to adhere for 90 min. Cell spreading was quantified as indicated in "Materials and Methods." Fig. 6ACitation shows the differential effects of this soluble paracrine factor on the spread of LNCaP and C4-2 cells. Although all conditioned media caused C4-2 cells to spread more rapidly on laminin, none had any noticeable effect on the spreading of LNCaP cells. No increase in spreading was seen for either cell line when treated with conditioned media from mouse fibroblastic cells (Sw3T3 cells; data not shown).



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Fig. 6. LNCaP and C4-2 cell attachment to laminin substrata after treatment with stromal cell-conditioned media and/or integrin isotype-specific antibodies. A, LNCaP and C4-2 cell attachment responses to conditioned media from four primary prostate stromal cell cultures (three different patients). All percentage attachment values are normalized to the behavior of control, LNCaP, and C4-2 cells that were not treated with conditioned media (after 90 min, 25% of C4-2 untreated control cells had attached, compared with only 6% of LNCaP untreated control cells). B, comparison of C4-2 cell attachment percentages after treatment with stromal cell-conditioned media in the presence or absence of function-blocking, integrin isotype-specific antibodies. All experiments were repeated six times. Statistically significant differences between treated and untreated cells were at P < 0.001 (*).

 
The effects of conditioned media could be reversed using integrin isotype-specific, function-blocking antibodies. Fig. 6BCitation shows that function-blocking antibodies to both {alpha}6 and ß1 inhibit cell spreading in both control and stromal cell-conditioned media-treated cells. Function-blocking antibodies against the {alpha}2, {alpha}3, and {alpha}vß3 integrins also were able to block 20–50% of the increase in cell spreading induced by conditioned media. Quantification of integrin cell surface expression by FACS analyses (Fig. 7)Citation did not reveal any change in receptor availability between control and stromal cell-conditioned media-treated cells, thus the observed variation in cell response to external regulation is unlikely to be based on changing integrin profiles but instead appears to be based on the improved efficiency of C4-2 cells in use of specific integrin isotypes for cell spreading in the presence of prostate stromal cell-conditioned media.



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Fig. 7. FACS analysis of cells treated with stromal cell-conditioned media. Surface expression of integrin subtypes after 90 min of treatment with stromal cell-conditioned media is shown. No change in the integrin surface expression is evident.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
During prostate cancer progression, changes occur not only in the microenvironments of cells but in the cells’ reception and interpretation of cues from these environments. We have focused here on shifts in integrin receptor expression and usage accompanying cancerous progression in the prostate and occurring in response to cues, such as stromally secreted factors. Although we did find expression levels of the integrin subunit {alpha}2 to be elevated in metastatic cell lines, overall, the usage of integrin subunits varied more strikingly than did expression level between cell lines and varied in response to exposure to stromal factors. The integrin usage we detected in the LNCaP model system correlated well with previously published immunohistochemical staining for integrin expression in patient specimens (Table 2Citation ; Refs. 5, 6, 7, 8 , 33, and 34 ) and added to a number of past in vitro studies showing differences in integrin heterodimer expression among cultured normal, neoplastic, and prostate carcinoma cells (10 , 34, 35, 36, 37) . Our results may also help clarify previous studies of integrin expression in various epithelial carcinomas, whose results have conflicted with one another, and may have implications in prostate cancer cell homing to the skeleton, along with preferential survival and proliferation in the bone microenvironment.


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Table 2 Comparison of previously reported integrin usage during prostate cancer progression and the usage found in the LNCaP progression model.

 
Integrin subunit partner choice repeatedly appeared to affect cell migratory and adhesive behaviors in the absence of shifts in subunit expression levels. For example, the partner choice of the integrin {alpha}6 subunit varied between cell lines, whereas the integrin ß1 and ß4 subunit expression levels remained constant among lines of very different invasive behaviors. LNCaP cells attached to laminin primarily with {alpha}6ß4, whereas cells in the more invasive C4-2 subline attached with {alpha}3ß1 and {alpha}vß3 (Figs. 2Citation and 3)Citation . This shift in {alpha}6 usage fits with previous studies, in which {alpha}6ß1 and {alpha}6ß4 were both found in normal prostate cells, but ß4 subunit expression was lost in carcinomas (5 , 6 , 8) . Because the {alpha}6 integrin subunit preferentially associates with ß4, it is believed that a reduction in ß4 subunit expression results in a relative increase in the formation of the {alpha}6ß1 heterodimer (38) . The varied pattern of integrin heterodimerization in these cell lines could be due in part to differential expression of {alpha}6 subunit isoforms. The integrin {alpha}6 subunit exists as two isoforms, {alpha}6A and {alpha}6B (34 , 38, 39, 40, 41, 42) , both of which are expressed in LNCaP cells (34) . Although we do not yet know the specific {alpha}6 isoform expressed by C4-2 cells, overexpression of the {alpha}6A isoform is known to increase {alpha}6ß1 heterodimerization as well as overall cell motility, tumorigenicity, and invasion (12) .

{alpha}6ß4 use declined in C4-2 cells, in conjunction with an increased use of {alpha}6ß1, {alpha}vß3, and {alpha}3ß1 (Fig. 2)Citation . Unlike {alpha}6ß4, which is associated with stable, hemidesmosomal cell attachment sites and appears to restrict cell migration, {alpha}6ß1 and {alpha}3ß1 are both involved in the formation of dynamic focal contacts important for cell locomotion (12) . Prostate cell lines able to form invasive tumors in immunocompromised mice have previously been shown to have increased expression of the {alpha}6ß1 heterodimer (10 , 12 , 36 , 43) , and antibodies against {alpha}6ß1 are able to inhibit invasion. Like Vafa et al. (36) , we too found the {alpha}6ß1 heterodimer to be more involved in cell spreading than static cell attachment (Figs. 2Citation and 6)Citation , but the role of this heterodimer in metastatic cell interpretation of environmental cues, such as matrix and secreted factors, requires further study. The {alpha}3ß1 integrin is likely to have both direct and indirect effects on cell motility because of its bidirectional interactions with the matrix. The ability of {alpha}3ß1 to alter laminin chains and overall basement membrane architecture (44 , 45) is particularly suggestive, given that proteolytic cleavage of laminin can drive cells from static adhesion to active migration (46 , 47) . It is interesting to note in this context that oncogene-transformed rat prostate cells express elevated levels of both laminin type I and the {alpha}6ß1 integrin (36 , 43) .

An additional integrin heterodimer implicated in increased metastatic potential and tumorigenicity is {alpha}vß3 (48, 49, 50) . Although not frequently found in epithelial cells, {alpha}vß3 is common to a number of bone-receding metastases, including prostate and breast carcinomas (10 , 11 , 51) . In the LNCaP model system, {alpha}vß3 was similar to {alpha}3ß1 in that its individual subunits were expressed at all stages of cancerous progression (that is, in all cell lines), but the assembled, functional heterodimers were only detectable in the more metastatic cell lines C4, C4-2, and C4-2B (Table 1Citation ; Fig. 3Citation ). Although such differences in integrin usage have been noted before between very different cell lines with different metastatic potentials, this is the first study we know of that reveals such shifts in integrin usage between cells with common genetic backgrounds but different in vivo metastatic potentials.

Two possible consequences of {alpha}vß3 heterodimer usage in the metastatic LNCaP sublines are (a) preferential relocation to the bone and (b) increased cell survival/suppressed cell death. Integrins are likely to be involved in both the establishment of prostate cell anchorage to the bone endothelium and its surrounding matrix and the transmission of multiple cues from the cells’ microenvironments supporting cell survival and proliferation. Not only do C4-2 cells, cells known to preferentially relocate to bone (26) , increase their use of the {alpha}vß3, but we show here that they use this integrin to migrate on OPN, a key component of bone matrix. {alpha}vß3 has also previously been shown to support migration on VN, another dominant component of bone matrix (52 , 53) . Regardless of the role of {alpha}vß3 in binding metastatic cells to the bone matrix, this integrin heterodimer is a good candidate regulator of cell survival in the absence of cell adhesion. Although loss of appropriate adhesion is normally a cue for apoptosis, human breast cancer cells are able to use {alpha}vß3 to inhibit p53 activity and suppress the bax death pathway (54) . Likewise, {alpha}vß3 has been shown to regulate cell proliferation in prostate epithelia (55) .

Integrin regulation of prostate epithelial proliferation is likely to involve interactions between integrins and growth factor receptors. Such receptors are used by cells to interpret positive and negative growth factor and cytokine signals from surrounding stromal cells (Refs. 1 and 56 and the references within), and they do so through common signaling cascade components (for example the small GTPases), which are also important for integrin signaling and activation. There is evidence that the two types of surface proteins may associate directly and preferentially with one another (55) . In the context of changing integrin usage [such as that observed between LNCaP and C4-2 cells or reported previously in vivo (see Table 2Citation )], preferential associations between the growth factor receptors and the changing integrin heterodimers could have serious consequences for the cells’ responses to environmental cues. Indeed, Fig. 6Citation shows that C4-2 and LNCaP cells do respond differently to soluble factors in media from prostate stromal cells, with only C4-2 cells showing increased spreading on laminin substrates after stromal media treatment.

We investigated the roles of {alpha}6ß1 and {alpha}vß3 integrins in response to stromal cues by adding function-blocking antibodies against these integrins to C4-2 cell cultures before and after treatment with prostate stromal cell-derived conditioned media. The dramatic increase in C4-2 laminin spreading after treatment with such conditioned media was relatively unaffected by {alpha}vß3 function-blocking antibodies, whereas antibodies against either {alpha}6 or ß1 completely obliterated spreading on laminin both before and after stromal cell-conditioned media treatment (Fig. 7)Citation , a result in agreement with the work of Vafa et al. (36) on c-erb B2/neu-transformed rat prostate epithelial cells. The identities of the responsible soluble factors (cytokines, growth factors, or others) behind the {alpha}6ß1-specific response remain to be determined, although one candidate growth factor, which is found in the conditioned media, is the HGF/SF.5 Although purified HGF/SF has the ability to stimulate prostate cancer cell spreading and migration when placed on ECM substrata, HGF/SF is only one of many such factors secreted by the prostate stromal cells. Whether growth factor receptors on the C4-2 cells interact directly with nearby {alpha}6ß1 integrins after stimulation by a stromally secreted growth factor is also unknown. Stimulated receptors could also signal the integrins indirectly through intracellular cofactors, such as focal adhesion kinase (62) .

In summary, use of a lineage-derived LNCaP cancer cell progression model has allowed us to compare the integrin expression levels, heterodimer usage, and cell behaviors in cells sharing a common genetic background but differing in their metastatic potentials on different matrices and in the presence or absence of stromal factors. We have found that although integrin expression levels do not change markedly among the cell lines (with the exception of an increase in collagen binding {alpha}2 expression), integrin heterodimer usage does change. In particular, the androgen-independent and invasive LNCaP derivative C4-2 subline shows marked differences in its use of {alpha}3ß1, {alpha}6ß1, {alpha}6ß4, and {alpha}vß3 when compared with that of the androgen-dependent and noninvasive parental LNCaP cells. Although all cells attached to laminin, VN, and OPN matrices, only the more invasive and metastatic C4-2 cells were able to migrate on OPN. C4-2 cells were also unique because of their response to prostate stromal cell-derived factors. The striking increase in the spreading of C4-2 cells on laminin after treatment with stromal factors could be completely obliterated by the addition of function-blocking antibodies against {alpha}6 or ß1, but not against {alpha}2, {alpha}3, ß4, or {alpha}vß3. Because C4-2 cells were found to increase usage of {alpha}6ß1 but decrease usage of the {alpha}6ß4 heterodimer, additional studies are called for to characterize this shift in heterodimer usage and its direct and/or indirect effects on cell behavioral and survival responses to matrix and stromal environmental cues. Such future studies promise to have profound implications for control of metastatic human prostate cancer cell dissemination, proliferation, and survival in the skeleton.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture, Antibodies, and ECMs.
LNCaP cells and their more metastatic sublines, C4, C4-2, and C4-2B (25) , were grown in T-media (Life Technologies, Inc., Rockville, MD) supplemented with 5% fetal bovine serum. Primary cultures of prostate stromal cells were derived from the tissue surrounding prostatic adenocarcinomas, as described by Ozen et al. (57) . Conditioned media were prepared by adding fresh media without serum when cells reached 80% confluence and removing it 48 h later. Laminin-1 was purified from Engelbreth-Holme-Swarm tumors according to Davis et al. (58) , based on the protocol of Kleinman et al. (59) . OPN-2 was purified as described by Devoll et al. (60) . VN was purchased from Promega (Madison, WI). Antibodies to integrin subunits {alpha}2, {alpha}3, {alpha}6, {alpha}vß3, ß1, ß3, and ß4 were all obtained from Chemicon (Emecula, CA). Vinculin antibody (V9131) was obtained from Sigma (St. Louis, MO), and all secondary-conjugated antibodies were obtained from Jackson Immunochemicals (West Grove, PA).

Immunofluorescent Confocal Microscopy.
Cells were seeded onto glass coverslips coated with 50 µg/ml laminin-1. For immunocytochemistry, cells were allowed to spread, fixed in 3% formaldehyde, permeabilized in 0.2% Triton X-100, and stained using either FITC-labeled phalloidin to label filamentous actin or antivinculin antibody (V9131) to detect focal adhesions. Texas Red-conjugated goat antimouse secondary antibodies were obtained from Jackson Immuno Research (Bar Harbor, ME). Cells were mounted on glass coverslips with gel-Mount (Biomedia Corp.), and images were acquired using a laser-scan confocal microscope 410 (Carl Zeiss, Minneapolis, MN).

Flow Cytometry Analysis.
Cells below 70% confluence were detached from tissue culture plates and suspended as single cells using a brief treatment of 10 mM EDTA and 20 mM HEPES buffer (pH 7.4) in T-media. The EDTA was neutralized with CaCl2 and MgSO4, and the cells were washed again with T-media containing 0.1% BSA. A total of 2.5 x 105 cells were used for each preparation. Cells and primary antibodies (30 µg/ml) were incubated for 60 min at 4°C, washed, and further incubated with secondary FITC-labeled goat antimouse (30 µg/ml) antibody for an additional 60 min at 4°C. After three brief washes, 1 x 104 cells were analyzed for fluorescence using a FACScan (Becton Dickinson, San Jose, CA). Cells treated with isotype-specific immunoglobulins served as controls. For both cell types, the relative fluorescence intensity was expressed as the increase over background fluorescence. Data points were presented as the mean of two independent experiments, with a range in parentheses (Table 1)Citation .

Substrate Adhesion, Attachment, and Migration Assays.
Cell attachment and competition assays were performed as described by Vafa et al. (36) . Assay plates were precoated with laminin, VN, or OPN by overnight incubation at 4°C and subsequently blocked with heat-inactivated BSA for an additional 4 h at room temperature. For adhesion assays, cells were trypsinized with 0.2% trypsin/2% EDTA in PBS (pH 7.2), suspended in T-media for titration to single cell suspension, and centrifuged briefly. Resuspended cells were then held in adhesion media [T-media with 20 mM HEPES (pH 7.4), 7 mM EDTA, and 0.1% BSA] for 5 h at 37°C and 5% CO2 to ensure reexpression of integrins on the cell surface. After preincubation, CaCl2 and MgSO4 were added to neutralize EDTA. Cells (5 x 103) in 100 µl of serum-free media were added to each well and allowed to attach for 6 h at 37°C. Triplicate cultures were prepared for each condition. After culture, cells were washed twice in PBS and stained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (61 , 62) or visually counted.

For the attachment assay with or without stromal cell-conditioned media, cell lines were grown to confluence, trypsinized, and replated (1:8) on tissue culture dishes, where they were allowed to grow for another 2 days before being lifted and treated briefly with 10 mM EDTA and 20 mM HEPES buffer in T-media. After neutralizing the EDTA with CaCl2 and MgSO4, the cells were washed with T-media containing 0.1% BSA. Cells were finally held in unconditioned or stromally conditioned media for 10 min, placed on laminin-coated dishes, allowed to adhere for 90 min, and then fixed in formaldehyde. Visible lamelopodia or fillopodia categorized a cell as positively spread. Each cell line was scored for the percentage of spread cells, and all values were normalized to that of control cells that had not been subjected to treatment with conditioned media. At 90 min, untreated LNCaP cells spread on laminin and VN at percentages of 5–10% and 45%, respectively, while at the same time point, untreated C4-2 cells spread on laminin and VN at percentages of 25–35% and 40%, respectively.

Haptotaxis was assayed in triplicate using modified Boyden chambers with an 8 µm pore size [Becton Dickinson (Bedford, MA) or Corning (Acton, MA)]. PBS (100 µl) containing laminin (50 µg/ml), VN (50 µg/ml), or OPN (20 µg/ml) was placed on the underside of the porous membrane and chambers were pre-incubated at 4°C overnight. PBS (100 µl) alone served as a negative control. On the second day, chambers were assembled with serum-free T-media containing 0.1% BSA. Cells (5 x 104 ) were added to the upper chambers and incubated at 37°C, 5% CO2 for 16 h. Cells were then fixed with 2% parafomaldehyde and stained with crystal violet. Cells remaining in the upper chamber were removed with a cotton swab. Cells that had migrated were counted using light microscopy; for each condition, 10 randomly chosen fields of cells were counted, and the results were presented as an average ± SD. Migrated control cells were counted at densities of approximately 100 cells/mm2.

Cell Surface Biotinylation.
Integrins on cells surfaces were biotinylated as described previously (63) . Briefly, cells were washed in PBS and incubated with 500 µg/ml sulfo-NHS-LC-biotin (Pierce, Rockford, IL) for 30 min at room temperature. Cells were then washed in 50 mM glycine and PBS before they were lysed [20 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP40, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin]. Cell extracts were precleared with protein A/G-agarose beads (Oncogen Science, Cambridge, MA) for 1 h at 4°C and spun at 10,000 rpm for 30 min. Integrin subunits were retrieved by immunoprecipitation. Integrin subunit-specific antibodies (200–500 µg/ml) were incubated with the cell lysate for 1 h at 4°C, and immunocomplexes recovered using protein A/G-coated agarose beads. Complexes were analyzed by nondenaturing 7.5% PAGE and electroblotting. After transfer, filters were blocked in 5% milk for 1 h at room temperature. Filters were then incubated with horseradish peroxidase-streptavidin, and proteins were detected using enhanced chemiluminescence (Amersham, Piscataway, NJ).

Statistical Analyses.
Where applicable, data were analyzed using Excell or QuickTTest, for determination of mean, SD, and parametric statistics (paired t test).


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Swedish Natural Science Research Council Grant B11479-300 and United States Department of Defense Grant PC990037 (to M. E.) and by NIH Grant CA-76620 and grants from the Kluge and CaPCURE Foundations (to L. W. K. C.). Back

2 M. E. and T. M. contributed equally to this study. Back

3 To whom requests for reprints should be addressed, at Department of Urology, Box 800422, Molecular Urology and Therapeutics Program, University of Virginia Health System, Charlottesville, VA 22908-0422. Phone: (804) 243-6649; Fax: (804) 243-6648; E-mail: Chung{at}virginia.edu Back

4 The abbreviations used are: ECM, extracellular matrix; FACS, fluorescence-activated cell-sorting; VN, vitronectin; OPN, osteopontin; HGF/SF, hepatocyte growth factor/scatter factor. Back

5 M. Edlund, T. Miyamoto, R. A. Sikes, R. Ogle, G. W. Laurie, M. C. Farach-Carson, C. A. Otey, H. E. Zhau, and L. W. K. Chung. Regulation of cell adhesion in prostate cancer cell lines by hepatocyte growth factor, manuscript in preparation. Back

Received for publication 10/27/00. Revision received 12/20/00. Accepted for publication 12/21/00.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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