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Cell Growth & Differentiation Vol. 10, 479-490, July 1999
© 1999 American Association for Cancer Research

Expression of the {alpha}7ß1 Laminin Receptor Suppresses Melanoma Growth and Metastatic Potential1

Barry L. Ziober2, Yao Qi Chen, Daniel M. Ramos, Nahid Waleh and Randall H. Kramer3

Departments of Stomatology [B. L. Z., Y. Q. C., D. M. R., R. H. K.] and Anatomy [R. H. K.], University of California San Francisco, San Francisco, California 94143; and SRI International, Menlo Park, California 94025 [N. W.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The {alpha}7ß1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of {alpha}7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar ß1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of {alpha}1 and abundant {alpha}6 but low or undetectable levels of {alpha}2 and {alpha}7. By contrast, C-23 cells expressed low or undetectable levels of {alpha}1, {alpha}2, and {alpha}6 but had up-regulated levels of {alpha}7. Consistent with the protein data, Northern blot analysis showed that levels of {alpha}7 mRNA were highest in the poorly metastatic variant cells, whereas {alpha}6 message was not detected; in contrast, {alpha}6 mRNA was elevated in the highly metastatic cells, whereas {alpha}7 message was not detected. Forced expression of {alpha}7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the {alpha}7ß1 laminin receptor.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The process of melanoma tumor growth and dissemination involves specific interactions with tumor cell surface adhesion receptors and multiple adhesive components of the ECM.4 For example, stationary cells form stable focal adhesions with the ECM, whereas motile cells form weak and transient contacts. The receptors that mediate the cellular adhesive interactions with the ECM are derived from a large family of heterodimeric molecules referred to as the integrins (1) . An abundance of evidence has shown that alterations in integrin expression are linked to tumorigenicity and metastasis (2 , 3) . In melanoma, several integrins, including the ß1 integrins and {alpha}vß3, have been implicated in the disease process (2 , 4, 5, 6) .

Some of the most pronounced changes in melanoma integrin expression occur in the laminin-binding receptors. Laminin contains multiple sites for cell attachment, which is mediated by several ß1 integrins. The major laminin receptors are {alpha}1ß1, {alpha}2ß1, {alpha}3ß1, and {alpha}6ß1 and are typically up-regulated during conversion to malignant melanoma (7, 8, 9, 10, 11, 12) . Increasing evidence indicates that integrin-laminin interactions not only promote cell attachment but can also stimulate cell migration, tumor growth, metastasis, angiogenesis, and protease production (13, 14, 15, 16) . Overall, the laminin integrin receptors appear to play some active role in the processes that lead to melanoma invasion and metastasis.

Previously, we reported the identification and characterization of the integrin complex designated {alpha}7ß1 (17, 18, 19) . More recently, we showed that this integrin complex is a laminin receptor that adheres to laminin-1 and laminin-2/4 (20) . Although originally identified as a laminin receptor in human and murine melanoma, {alpha}7ß1 integrin has been primarily characterized in muscle (17 , 21, 22, 23, 24) . In skeletal and smooth muscle, {alpha}7 expression is regulated in a differentiation specific manner, being highly expressed in the terminal differentiated nonproliferated state (17 , 22, 23, 24) . The function of {alpha}7 in melanoma has yet to be defined.

The murine K1735 melanoma model is a well-characterized tumor system that is composed of several clones that differ in tumorigenicity and metastatic potential (25 , 26) . In this study, we examined the role of laminin-binding integrins in metastatic and nonmetastatic cell lines derived from the parental K1735 cells. We show that, in the K1735 melanoma variants, there is an inverse correlation between expression of the {alpha}7 subunit and metastatic potential. Transfection of the {alpha}7 subunit into {alpha}7-null metastatic cells resulted in a cell line that was less motile and metastatic and formed smaller tumors. Our results suggest that loss of {alpha}7ß1 during melanoma progression contributes to the tumorigenic and metastatic phenotype.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Adhesion and Migration of Melanoma Cell Variants on Laminin-1 and E8 Fragment.
In adhesion assays, we found that both the highly tumorigenic and metastatic M-2 and the less tumorigenic, poorly metastatic C-23 cells adhered well to laminin-1 (Fig. 1, A and B)Citation . Both cell lines reached maximal binding at {approx}30 µg/ml laminin. In addition, both M-2 and C-23 cells adhered to the E8 fragment, which represents the long arm of the laminin molecule. However, C-23 cells bound the E8 fragment more efficiently at lower coating concentrations than they bound laminin (Fig. 1A)Citation , and they bound more efficiently than did the M-2 cells (Fig. 1B)Citation . Blocking antibodies specific to ß1 integrins and to the E8 fragment could separately block adhesion of C-23 cells to laminin and E8, respectively (Fig. 1C)Citation . M-2 cell binding to laminin and E8 was also inhibited with anti-ß1 blocking antibodies, but anti-E8, although able to disrupt binding to E8, only partially blocked binding to laminin (Fig. 1D)Citation . Thus, in both cell lines, ß1 integrins, in particular those interacting with the E8 fragment of laminin, are responsible for adhesion to laminin-1.



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Fig. 1. Adhesion and migration of K1735 melanoma cell lines. A and B, dose response of adhesion of C-23 (2 x 104/well; A) and M-2 cell (2 x 104/well; B) to laminin-1 ({blacksquare}) and the E8 fragment of laminin (•). C and D, C-23 cells (2 x 104/well; C) and M-2 cells (2 x 104/well; D) were tested for adhesion to laminin-1 (at 10 µg/ml; {square}) or laminin E8 (at 5 µg/ml; ), as described in "Materials and Methods." Blocking antibodies to ß1 (AIIB2) or to the E8 fragment of laminin or control IgG were preincubated with cells at 10 µg/ml. For all adhesion assays, cells bound to collagen type I (at 100 µg/ml) were used to indicate 100% adhesion. Adherence of cells in 1% BSA-coated wells was treated as background binding and subtracted. A–D, data are presented as percentages of the total cells added to each well. Data points (A and B) and columns (C and D), means of triplicate wells; bars, SD. E, dose response of M-2 (•) and C-23 ({blacksquare}) cell migration on laminin-1-coated surfaces. F, migration of M-2 ({blacksquare}) and C-23 () cells on surfaces coated with laminin-1 (10 µg/ml), the laminin E8 fragment (5 µg/ml), and fibronectin (20 µg/ml). Migration was measured using the microscreen assay, as described in "Materials and Methods." The area covered by out-migrating cells, from the fixed diameter of the microscreen, was measured by computer-assisted image analysis (NIH Image). Data points (E) and columns (F), means of at least five individual measurements in pixel units (x 103) and represent "relative migration"; bars, SD.

 
In contrast to adherence, migration on laminin-1 was substantially different for M-2 and C-23 cells. The highly metastatic M-2 cells migrated efficiently over a wide range of laminin-1 concentrations, whereas C-23 cells migrated poorly or not at all (Fig. 1E)Citation . For both cell lines, optimal migration occurred at coating concentrations of 10–30 µg/ml; at higher concentrations, migration was inhibited (Fig. 1E)Citation . The E8 fragment as ligand was also able to induce a differential motility response, with the M-2 cells moving more rapidly on E8 than the C-23 cells (Fig. 1F)Citation . On fibronectin, the migration efficiencies were reversed: the M-2 cells migrated slightly more slowly than the C-23 cells (Fig. 1F)Citation , indicating that the poor migration of C-23 cells on laminin-1 is not due to a general defect in their migratory activity. Thus, the fact that M-2 and C-23 cells require ß1 integrins for adhesion on laminin-1 and E8 suggests that these same ECM receptors are also responsible for the contrasts in cell migration.

Integrin Cell Surface Profile.
To determine which ß1 integrins were responsible for adhesion and, thus, migration on laminin-1, we performed Western immunoblotting. We previously showed that {alpha}7ß1 is expressed in several melanoma cell lines and that it binds primarily to laminin-1 (4 , 18 , 19) . By Western blot analysis, the two poorly metastatic cell lines, C-23 and C-19, expressed high levels of the {alpha}7 subunit (Fig. 2A)Citation . In contrast, the M-2 cells expressed little to no {alpha}7. These initial results suggested that differentially expressed integrin receptors may be responsible for the adhesive and migratory properties displayed by these cells on laminin. Therefore, we analyzed the integrin profiles of M-2 and C-23 cells further.



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Fig. 2. Expression of {alpha}7 mRNA in K1735 cell variants. A, equal quantities of cellular lysates from M-2 (Lane 1), C-19 (Lane 2), and C-23 (Lane 3) cells were processed for SDS-PAGE in a 7.5% polyacrylamide gel. Following transfer, the nitrocellulose membrane was probed with anti-{alpha}7 (1211) antiserum and the position of the {alpha}7 subunit was determined using the color development detection system for AP. The position of the {alpha}7 subunit is indicated. The band at Mr {approx}70,000 is a proteolytic fragment of the {alpha}7 subunit (20 , 24) . B, total RNA was isolated as described in "Materials and Methods" from nonmetastatic (C-10, C-19, and C-23) and highly metastatic (C-26, M-2, and M-4) K1735 cell lines and analyzed by Northern blotting using a 32P-labeled murine {alpha}6 cDNA probe (a). The blot was stripped and then reprobed with an {alpha}7 cDNA probe (b). Positions of the {approx}6.0-kb {alpha}6 mRNA and the {approx}4.1-kb {alpha}7 mRNA transcripts are indicated. Equal loading was assessed by ethidium bromide staining.

 
We used FACS analysis to examine the cell surface integrin profile for M-2 and C-23 cells (Table 1)Citation . Both cell lines expressed very little {alpha}2. In M-2 cells, {alpha}6 and {alpha}1 were the most prominent laminin-1-binding integrin subunits expressed. Little {alpha}7 was detected in M-2 cells. In contrast, C-23 cells expressed high levels of {alpha}7 and very little {alpha}6 or {alpha}1. Thus, in the highly metastatic M-2 cells, {alpha}6 and {alpha}1 appear to be the major laminin-1 receptors, whereas {alpha}7 is the major laminin-1 receptor in the nonmetastatic C-23 cell line. These results also suggest that, during conversion to the highly tumorigenic/metastatic melanoma phenotype, {alpha}7 expression is down-regulated and that of {alpha}1 and {alpha}6 is up-regulated. We, therefore, hypothesized that the {alpha}7 subunit may play a role in regulating cell motility and, thus, metastatic potential.


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Table 1 Flow cytometry-determined expression levels of laminin-binding integrin subunits in K1735 M-2, C-23, and M-2-{alpha}7 cell lines

 
Gene Transcription.
To identify a mechanism responsible for the differential expression of the {alpha}6 and {alpha}7 proteins in M-2 and C-23 cells, we performed Northern blot analysis. We screened total RNA from a panel of K1735 melanoma cell lines with differing metastatic potential, using cDNA probes for {alpha}6 and {alpha}7. The mRNA levels for {alpha}6 were consistently elevated in cells of high metastatic potential (C-26, M-2, and M-4) but low or undetectable in cell lines that were poorly metastatic (C-10, C-19, and C-23; Fig. 2BCitation ). In contrast, mRNA levels for {alpha}7 reflected those seen for protein expression: high in poorly metastatic cell lines like C-23 and undetectable in the highly metastatic cell lines like M-2 (Fig. 2B)Citation . By densitometric analysis, the relative level of {alpha}7 transcripts in C-23 cells was at least 10-fold that of the M-2 cells (data not shown). Thus, the highly metastatic cells lacked expression, at both the protein and mRNA levels, of the laminin-binding integrin complex {alpha}7ß1.

Forced Expression of {alpha}7 in Metastatic Cells.
To directly determine whether {alpha}7ß1 plays a role in regulating melanoma motility and metastatic potential, we transfected the full-length {alpha}7 cDNA into M-2 cells by retroviral infection. After selection with G418, a population of {alpha}7-expressing M-2 cells (designated M-2-{alpha}7) were isolated after two rounds of FACS analysis (Fig. 3)Citation . Expression of {alpha}7 in M-2 parental and M-2-{alpha}7 cells was analyzed by Western blotting (Fig. 4)Citation . The level of {alpha}7 in M-2-{alpha}7 cells exceeded that expressed by C-23. Upon reduction, {alpha}7 expressed in M-2-{alpha}7 cells displayed the characteristic light chain (Mr 37,000, representing the cytoplasmic domain; Ref. 20 ; Fig. 4Citation ). Western blotting detected little to no {alpha}7 expression by M-2 parental cells (Fig. 4)Citation . However, some expression of {alpha}7 could be seen in the M-2 parental cells when the more sensitive method of FACS analysis was used (Table 1)Citation . This agrees with our observation that {alpha}7 protein can be seen by Western blots in M-2 cells only after prolonged exposure to X-ray film or protein overloading (data not shown). M-2 cells expressed at least 5-fold less {alpha}7 than the nonmetastatic C-23 cells, as determined by FACS (Table 1)Citation . In contrast, {alpha}7 levels in M-2-{alpha}7 cells were nearly 10-fold higher than levels in the M-2 parental cells and 2-fold higher than C-23 cells. Thus, the transfected M-2 cells now expressed {alpha}7 at levels similar to those of the nonmetastatic C-23 cell line. Furthermore, the FACS analysis confirmed that this expression was located at the cell surface.



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Fig. 3. Analysis of {alpha}7 cell surface expression in M-2, M-2-{alpha}7, and Mock cell lines. Flow cytometry analysis of the cell-surface expression levels in M-2, M-2-{alpha}7, and Mock cell lines was performed with optimal concentration of mAb CY8 (anti-{alpha}7), followed by incubation with FITC-labeled goat antirat IgG. The retrovirally transfected cell lines M-2-{alpha}7 and Mock were sorted for expression of {alpha}7 and lack of {alpha}7 expression, respectively. As a control, cells were stained with secondary antibody only (2° Antibody). {alpha}7 expression peaks for each cell line are indicated.

 


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Fig. 4. Expression of {alpha}7 in M-2, M-2-{alpha}7, and C-23 cells. Equal quantities of cellular lysates from M-2 (Lanes 1 and 4), M-2-{alpha}7 (Lanes 2 and 5), and C-23 (Lanes 3 and 6) cells were processed for SDS-PAGE in a 7.5% polyacrylamide gel under nonreducing (Lanes 1–3) and reducing (Lanes 4–6) conditions. Following transfer, the nitrocellulose membrane was probed with anti-{alpha}7 (1211) antiserum and the position of the {alpha}7 subunit was determined by ECL. When reduced, the cytoplasmic domain of the {alpha}7 subunit is released (20) . The positions of the {alpha}7 subunit and the released cytoplasmic domain are indicated.

 
Because alterations in integrin expression are linked to motility, differentiation, cell attachment, tumor growth, and metastasis (2 , 3) , we asked what changes in integrin expression occurred as a consequence of {alpha}7 expression in M-2 cells. Forced expression of {alpha}7 resulted in no changes in {alpha}2 and only a modest decrease in {alpha}1 expression ({approx}2-fold reduction; Table 1Citation ). The most dramatic changes were seen in the expression levels of {alpha}7 and {alpha}6 (above and Table 1Citation ). Thus, expression of {alpha}7 in M-2 cells resulted in a cell surface integrin profile that now more closely resembled that of the nonmetastatic C-23 cell line (Table 1)Citation , in that {alpha}7 levels were high and {alpha}6 levels were reduced.

Adhesion to Laminin-1.
We next compared the laminin-1-binding ability of the M-2-{alpha}7 cells with that of M-2 parental and C-23 cell lines. All three cell lines adhered to laminin-1, but the M-2-{alpha}7 cells adhered more readily (Fig. 5)Citation . Anti-{alpha}2 antibodies did not block laminin-1 adhesion in any of the cell lines tested (data not shown). This probably reflects the fact that M-2, M-2-{alpha}7, and C-23 cells express little {alpha}2 (Table 1)Citation . Addition of blocking antibodies to {alpha}1 only slightly reduced the binding of any of the cell lines to laminin-1. When blocking antibodies to {alpha}6 were added alone, the adhesion of M-2 cells was reduced (Fig. 5A)Citation , indicating that the primary laminin receptor in the M-2 cell line is {alpha}6. In contrast, adhesion of C-23 and M-2-{alpha}7 cells was essentially unaffected by blocking antibodies to {alpha}6 or by a mixture of anti-{alpha}1 and -{alpha}6 (Fig. 5, B and C)Citation . Blocking antibodies to {alpha}7 slightly blocked M-2 cells but nearly completely inhibited M-2-{alpha}7 cells (Fig. 5D)Citation . Finally, when blocking antibodies to {alpha}7 were added to the anti-{alpha}1 and -{alpha}6 mAb mixture, adhesion to laminin-1 was completely inhibited for all cell lines. Thus, these results indicated that the contribution of {alpha}3ß1 to laminin-1 adhesion is minimal, if at all, in these cell lines, which is consistent with the laminin-5 isoform specificity of this integrin (27, 28, 29) . More importantly, these results demonstrate that M-2-{alpha}7 cells, in contrast to M-2 cells, use {alpha}7ß1 as their primary laminin-1 receptor.



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Fig. 5. Adhesion of M2, M-2-{alpha}7, and C-23 cell lines to laminin-1. Parental M-2 (A), C-23 (B), and M-2-{alpha}7 (C) cells and parental M-2 ({blacksquare}) and M-2-{alpha}7 () cells (D; 2 x 104/well) were tested for adhesion to laminin-1 (at 15 µg/ml) alone or in the presence of anti-integrin antibodies as described in "Materials and Methods." Ln, adhesion to laminin-1 only. Blocking antibodies to {alpha}1 (Ha 31/8), {alpha}6 (GoH3), and {alpha}7 (CY8) were preincubated with cells at 10 µg/ml. Cells bound to collagen type I (at 100 µg/ml) were used to indicate 100% adhesion. Adherence of cells in 1% BSA-coated wells was treated as background binding and subtracted. Data are presented as percentages of the total cells added to each well. Columns, means of triplicate wells; bars, SD.

 
Tumor Growth in Vivo.
Previous work has shown that, when M-2 cells are injected s.c. into syngeneic mice, they form aggressively growing tumors that are highly metastatic (25 , 30 , 31) . In contrast, the {alpha}7-expressing C-23 cell line forms slower-growing tumors that metastasize at lower efficiency, if at all (25 , 30 , 31) . Furthermore, {alpha}7 expression is associated with the terminally differentiated nonproliferating state in muscle. We, therefore, investigated whether increased expression of {alpha}7 in M-2 cells can affect in vivo tumor growth. M-2 parental and M-2-{alpha}7 cells were injected s.c. into syngeneic C3H/HeN mice. In all mice injected with M-2 parental cells, rapidly growing tumors were observed (Table 2Citation and Fig. 6Citation ). In contrast, mice injected with M-2-{alpha}7 cells showed reduced tumor take, and the slow-growing tumors that did form were, on average, at least 2-fold smaller, by size, than the M-2 parental cell tumors (Table 2Citation and Fig. 6Citation ). For example, by day 34, tumors produced from the M-2 cells were >41 mm in diameter or 38.8 x 103 mm3 in volume, whereas tumors derived from M-2-{alpha}7 cells did not exceed 23 mm in diameter or 6.4 x 102 mm3 (Table 2)Citation . Growth of M-2-{alpha}7 tumors started to plateau at day 29, a pattern that was more pronounced by day 34. In contrast, tumors established from M-2 parental cells were in logarithmic growth at day 29 and continued in logarithmic growth even up to day 34 (Table 2Citation and Fig. 6Citation ). At termination of the experiment, tumors originating from M-2 cells weighed {approx}20.0 g, on average, whereas those originating from M-2-{alpha}7 cells averaged only {approx}9.0 g. Expression of {alpha}7 was detectable, by Western blot analysis, in tumors derived from the M-2-{alpha}7 cells but not from tumors produced by M-2 cells (Fig. 7)Citation . Tumors originating from both cell lines were similar histologically.


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Table 2 Tumorigenicity of K1735 M-2 and M-2-{alpha}7 cell lines injected s.c.

 


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Fig. 6. Growth of M-2 and M-2-{alpha}7 cells in the subcutis of C3H/HeN mice. Tumor cells (4 x 105) in HBSS were injected s.c. into syngeneic C3H/HeN mice. Tumors produced from M-2 (•) and M-2-{alpha}7 cells ({blacksquare}) were monitored at the indicated days as described in "Materials and Methods." Ordinate, average tumor diameter (mm), means of five to six s.c. tumors; bars, SD.

 


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Fig. 7. Expression of {alpha}7 in tumors derived from M-2 and M-2-{alpha}7 cells. Equal quantities of cellular lysates from tumors derived from M-2 (Lane 1) and M-2-{alpha}7 (Lane 2) cells were processed for SDS-PAGE in a 7.5% polyacrylamide gel. Following transfer, the nitrocellulose membrane was probed with anti-{alpha}7 (1211) antiserum and the position of the {alpha}7 subunit was determined by ECL. The position of the {alpha}7 subunit is indicated ({alpha}-7).

 
As an added control, tumors derived from a population of retrovirally infected G418-resistant M-2 cells that do not express {alpha}7 (designated Mock; Fig. 3Citation ) were compared with tumors generated by M-2-{alpha}7 cells (Table 3)Citation . The results were very similar to those obtained with M-2 parental and M-2-{alpha}7 cells (Table 2)Citation . Mock M-2 tumors were faster-growing and nearly 2-fold larger than tumors generated by the {alpha}7-transfected M-2 cells. In particular, by day 21, tumors derived from Mock M-2 cells were >21 mm in diameter, whereas tumors from M-2-{alpha}7 cells did not exceed 12.5 mm in diameter (Table 3)Citation .


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Table 3 Tumorigenicity of K1735 Mock and M-2-{alpha}7 cell lines injected s.c.

 
Although the retroviral vector pLNCX-4 used to infect the M-2 cells was designed not to express any viral genes, so as to be usable for gene therapy, it was still possible that activation of the host immune system in the C3H/HeN mice was interfering with growth of the M-2-{alpha}7-generated tumors (25 , 32) . To determine whether augmented antigenicity was responsible for the observed differences in tumorigenicity of the M-2 parental and M-2-{alpha}7 cells, we repeated the experiments above using athymic nude mice (Table 2)Citation . Except for the observation that all tumors grew faster in the athymic mice, the results were very similar to those obtained in the normal syngeneic mice. M-2 parental cells tumors were faster-growing and, on average, 2-fold larger than tumors generated by the {alpha}7-transfected M-2 cells. In particular, by day 16, tumors derived from M-2 cells were >27 mm in diameter, whereas tumors from M-2-{alpha}7 cells did not exceed 14 mm in diameter (Table 2)Citation . At termination of the experiment, tumors originating from M-2 cells weighed 3.9 g whereas those originating from M-2-{alpha}7 cells averaged only 1 g.

Reduced Migration and Metastasis.
Because migration is the hallmark of an invading and metastasizing melanoma cell, we asked whether transfection of {alpha}7 into M-2 cells had any effect on their highly migratory phenotype (33 , 34) . Transfection of {alpha}7 reduced migration of M-2-{alpha}7 cells on laminin-1, as compared to the nontransfected M-2 cells, by nearly 2-fold. However, M-2-{alpha}7 cells still migrated 2-fold better on laminin-1 than the poorly migratory C-23 cells (Fig. 8A)Citation . The diminished migratory phenotype shown by the M-2-{alpha}7 cells suggested that transfection of {alpha}7 may also alter the cells’ metastatic potential.



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Fig. 8. Production of experimental lung metastasis by M-2 and M-2-{alpha}7 cells. A, migration of M-2 (), M-2-{alpha}7 ({blacksquare}), and C-23 ({square}) cells on laminin-1-coated surfaces. Migration was measured using the microscreen assay as described in "Materials and Methods." The area covered by out-migrating cells, from the fixed diameter of the microscreen, was measured by computer-assisted image analysis (NIH Image). Columns, means of at least five individual measurements in pixel units (x 103) and represent "relative migration"; bars, SD. B, M-2 () and M-2-{alpha}7 ({blacksquare}) cells (1 x 105) were injected into the lateral tail vein of syngeneic C3H/HeN mice (six mice per cell line per experiment). After 2 weeks, mice were sacrificed, and the number of experimental lung metastases was determined as described in "Materials and Methods." Columns, mean number of tumors per mouse; bars, SD. The results for M-2-{alpha}7 cells are significantly different from those for the nontransfected M-2 parental cell line by Student’s t test (P = 0.025).

 
It was previously shown that C-23 cells produce very few experimental pulmonary metastases, averaging only five lung nodules, whereas M-2 cells produce, on average, at least 250 experimental pulmonary metastases when 2 x 105 cells are injected (25 , 31) . To determine whether expression of {alpha}7 affected the metastatic potential of the M-2 cells, we injected 1 x 105 M-2 or M-2-{alpha}7 cells i.v. into syngeneic C3H/HeN mice. After 2 weeks, the mice were sacrificed, and the number of lung-tumor colonies was determined. All animals injected developed experimental metastases. However, on average, M-2-{alpha}7 cells produced >3-fold fewer lung tumors than the M-2 parental cells (Fig. 8B)Citation . Together, these results demonstrated that M-2-{alpha}7 cells were now intermediate between M-2 and C-23 cells with respect to reduced laminin motility and in vivo tumor growth and decreased metastatic potential.

Growth Properties.
Because M-2-{alpha}7 cells showed decreased in vivo tumor growth, we next asked whether transfection of {alpha}7 into M-2 cells had any effect on the in vitro growth properties of these cells. We first determined whether there was any difference in the growth of M-2 and M-2-{alpha}7 cells when seeded on plastic as compared to laminin-1. The results (Fig. 9A)Citation showed that the growth rates of M-2 and M-2-{alpha}7 cells on each substrate were similar. However, their growth rates on laminin-1 were slightly slower than their growth rate on plastic, but there was no difference in growth between M-2 and M-2-{alpha}7 cell lines.



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Fig. 9. In vitro proliferation properties of M-2 and M-2-{alpha}7 cells. A, proliferation rates of M-2 (•, M-2 on laminin-1; {blacktriangleup}, M-2 on plastic) and M-2-{alpha}7 ({blacksquare}, M-2-{alpha}7 on laminin-1; {blacktriangledown}, M-2-{alpha}7 on plastic) cells when plated either on plastic or laminin-1-coated dishes. For laminin-1, microtiter plates (96-well Immulon plates) were precoated with 10 µg/ml of laminin-1 in PBS for 1 h at 37°C in a humidified atmosphere. Tissue culture-treated microtiter plates were used for growth on plastic. Cell lines were plated at a density of 50 cells per well. Data points, average cell number from triplicate wells, determined on the indicated days; bars, SD. O.D., absorbance. B, proliferation rate of M-2 ({square}) and M-2-{alpha}7 () cells when plated in DMEM H16 containing either 0.5% or 10% FBS as described in "Materials and Methods." Columns, average proliferation rates from triplicate dishes, determined after 5 days of incubation at 37°C; bars, SD.

 
Next, we asked whether transfection of {alpha}7 into the M-2 cell line altered the cells’ dependence on serum growth factors. Growth in 0.5 or 10% serum had no effect on the proliferative capacity of M-2 as compared to M-2-{alpha}7 cells, except that both cell lines grew more slowly when plated in reduced serum (Fig. 9B)Citation . Finally, we found that there was no significant difference in growth rate when the transfected cells were grown on other ECM ligands, collagen and fibronectin (data not shown). Together, these results indicate that transfection of {alpha}7 into M-2 cells had no detectable effect on the in vitro growth properties of these cells.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we examined the role of laminin-binding integrin receptors in regulating the invasive behavior of melanoma cells. The well-characterized murine K1735 melanoma model, originally derived from a primary tumor, is composed of a mixture of phenotypically different clones with metastatic heterogeneity (26) . Our findings show that {alpha}7 integrin is poorly expressed in a set of highly tumorigenic and metastatic cell lines but is strongly expressed in nonmetastatic cells. This initial result suggested that {alpha}7 may function in regulating cell motility and metastatic potential. To define the function of {alpha}7 in metastatic melanoma, we overexpressed the receptor in the M-2 cell line. Our results show that forced expression of {alpha}7 in M-2 cells diminished the highly migratory phenotype and suppressed tumor growth and metastatic potential. This is the first reported example of reduced in vivo growth and decreased metastatic potential by transfection of a laminin-binding integrin receptor in metastatic melanoma.

The highly metastatic M-2 cells migrated efficiently on laminin-1, whereas the poorly metastatic C-23 cells exhibited little motility on the same ligand. Laminin-1 induced a strong haptotatic response in the metastatic M-2 cells but not in the C-23 cells, and a relatively narrow range of coating concentrations promoted locomotion. Palecek et al. (35) recently showed that cell migration is dependent on several factors, including substratum ligand levels, ligand-binding affinity, and cell integrin expression levels. These conclusions are consistent with our results described here; that is, a minimum density of laminin-1 was needed for attachment and subsequent motility, but, at higher concentrations, movement was suppressed. These migration-dependent factors may also account for the reduced migratory phenotype seen when {alpha}7 was overexpressed in M-2 cells. Whether reduced migration was due to differences in laminin-1 affinity between {alpha}6 and {alpha}7 or to an increase in the total laminin-1-binding receptors is unknown. However, we previously showed that {alpha}7 binds with more avidity to laminin-1 affinity columns than does {alpha}6 (4 , 18 , 19 , 36) .

A connection between laminin receptor expression and metastatic potential is illustrated by the cells’ ability to migrate. M-2 cells, which are both highly migratory and metastatic, use {alpha}6ß1 as their primary laminin receptor. In contrast, C-23 cells, which are nonmetastatic and poorly migratory, use {alpha}7 as their main laminin receptor. When the primary laminin receptor in M-2 cells was switched from {alpha}6ß1 to {alpha}7ß1 by overexpression of the {alpha}7 subunit, the migratory phenotype and metastatic potential were reversed. Importantly, the M-2-{alpha}7 cells still expressed relatively moderate levels of {alpha}1 and {alpha}6 laminin-binding receptors, but these integrins were unable to affect the motility-reducing properties of {alpha}7. These results indicate that, in murine melanoma, high-level expression of {alpha}7 can regulate the cells’ migratory properties. However, Echtermeyer et al. (37) reported that low-level expression of transfected {alpha}7 in a human melanoma cell line slightly increased laminin migration. In addition, we have seen that {alpha}7ß1 expression in other cell types, including smooth muscle cells, skeletal myoblasts, and {alpha}7-transfected MCF-7 breast carcinoma cells, can promote cell migration on laminin-1 substrates (20, 21, 22) . Whether these differences in {alpha}7-mediated cell motility are due to the total number of laminin-1 receptors expressed, the level of {alpha}7 expression, or cell type-specific factors has not been determined.

Support for the idea that {alpha}7 reduces melanoma cell migration and metastasis comes from the differential ability of {alpha}7 and {alpha}6 to bind immobilized laminin. We previously showed that {alpha}6 is eluted from a laminin-Sepharose column with 50 mM NaCl (4 , 18 , 19 , 36) . In contrast, {alpha}7 is strongly bound to laminin-Sepharose columns and can usually be eluted only by chelation of divalent cations (4 , 18 , 19 , 36) . We favor the possibility that melanoma cells expressing {alpha}6 as the major laminin receptor may bind laminin less efficiently, thereby facilitating motility and invasion. In contrast, those tumor cells expressing high levels of {alpha}7 may bind laminin so efficiently that motility and invasion are suppressed. It is interesting that, in somatic cell hybrid analyses, fusion of M-2 cells and {alpha}7-expressing C-23 cells resulted in hybrids that were dramatically less metastatic (31) . Whether this is due to retention of the {alpha}7 subunit in the M-2/C-23 fusion clone or to other factors has yet to be determined. Finally, we recently established cell lines from rare lung tumors derived from C-23 cells. In these cell lines, little {alpha}7 expression was detected, supporting the notion that loss of {alpha}7 is required for metastasis (data not shown).

An important question arising from this study is why forced expression of {alpha}7 in the highly metastatic cells decreases tumor growth. A partial explanation may be related to the normal tissue expression pattern of this subunit. High {alpha}7 levels are primarily observed in skeletal, cardiac, and smooth muscle, where its expression is linked to the differentiation program (17 , 21 , 23 , 24 , 38) . At present, little is known about the regulation of {alpha}7 during cardiac muscle differentiation. However, in the terminally differentiated, nonproliferative state of skeletal and smooth muscle, {alpha}7 is highly expressed (17 , 21 , 23 , 24 , 38) . In contrast, during proliferation of skeletal muscle precursors or dedifferentiation of smooth muscle, {alpha}7 expression is lost (17 , 21 , 23 , 24 , 38) . These observations suggest that expression of {alpha}7 either is associated with or induces a nonproliferative, terminally differentiated state. Thus, in the low serum and plasma environment of the mouse, {alpha}7 may alter the proliferative or differentiation program in M-2-{alpha}7 cells, slowing their growth. Preliminary work has indicated that, when M-2-{alpha}7 cells are maintained in a serum-free environment, they fail to proliferate. In contrast, M-2 cells continue to proliferate but at a much reduced rate. Finally, when the mitogen-activated protein kinase pathway, which is primarily involved in cell proliferation, is inhibited in B16 melanoma cells, cell differentiation follows (39) . Whether expression of {alpha}7 in melanoma regulates the mitogen-activated protein kinase pathway or activates an antagonist pathway that may moderate cell proliferation or differentiation remains to be determined.

Several laminin integrin receptors, including {alpha}2ß1, {alpha}3ß1, and {alpha}6ß1, have been implicated in melanoma cell progression (7, 8, 9, 10, 11, 12) . Increasing evidence suggests that {alpha}6ß1 is a major laminin receptor in metastatic melanoma. Several studies have reported that {alpha}6 is frequently up-regulated in metastatic melanoma lesions (4 , 9 , 11) . Furthermore, recent work has shown that addition of laminin peptides can stimulate melanoma invasion in vitro and in vivo (40, 41, 42) . In particular, Nakahara et al. (42) showed that laminin peptides that interact with {alpha}6ß1 can induce invasion independently of {alpha}6’s adhesive functions. The interaction of {alpha}6ß1 with laminin appears to be required for the stimulation of invadopodia and extravasation during hematogenous metastasis; blocking antibodies to the {alpha}6 receptor can abolish experimental pulmonary metastasis (11 , 42, 43, 44) . Other studies have shown that, when ligated to laminin, the {alpha}6ß1 receptor can stimulate mitogenic activity that is independent of growth factor association (45) . We have shown here that, in M-2 cells, {alpha}6 plays a prominent role in adhesion to laminin. In addition, the M-2 parental cells form faster-growing tumors than M-2 cells overexpressing {alpha}7. Together, the results presented here and the observations above suggest that expression of the {alpha}6 laminin-binding receptor correlates not only with melanoma tumor growth but also with an invasive and metastatic phenotype.

In addition to {alpha}6, both {alpha}2 and {alpha}3 integrins are frequently up-regulated in melanoma. However, in K1735 cells {alpha}2ß1 was barely detectable and did not contribute to laminin adhesion. {alpha}3ß1 has been described as a receptor for several ECM ligands, including laminin-1, fibronectin, collagen, and thrombospondin (30 , 46, 47, 48) . More recent studies indicate that {alpha}3 is a receptor specific for laminin-5 (30 , 49) . In case of the {alpha}3ß1 receptor, our data do not support an important role for it in murine melanoma adhesion to laminin-1.

It is well established that tumor formation and acquisition of the metastatic state require a number of complicated processes and factors. Integrins appear to be important for several components of metastasis, including growth, invasion, and vascular dissemination. For example, several studies have implicated integrins in cell cycle progression and regulation of apoptosis (50 , 51) . Whether {alpha}7 has any effects on these processes in regard to regulating in vivo growth and metastasis is unknown. It has recently been suggested that the reduced tumor growth that follows transfection of {alpha}3 into rhabdomyosarcoma cells (52) may be the consequence of several altered parameters, including changes in ECM-ligand-binding interactions, secreted proteases (53) , and/or responsiveness to growth factors (54) . Any of these factors may contribute to the decreased tumor growth and metastatic potential we observed in the M-2-{alpha}7 cells. Finally, we showed previously that {alpha}7 has the greatest amino acid homology with {alpha}3 and {alpha}6 (17) . Both {alpha}3 and {alpha}6 have been shown to associate with members of the tetraspan family of molecules (55 , 56) . Such associations can alter the adhesive, proliferative, and migratory properties of these {alpha} subunits (55, 56, 57, 58, 59, 60) . Any involvement of {alpha}7 with members of the tetraspan family has not been reported.

The development of metastases, a highly selective process, is dependent on the existence of tumor cell variant subpopulations; that is, certain subsets of cells acquire a phenotype enabling them to survive the rigors of the metastatic cascade and successfully establish secondary foci. A successful phenotype would likely have a suitable array of integrin receptors that directly determine the cell’s adhesive and migratory activity. In the K1735 variant cell lines, the expression pattern of the {alpha}6ß1 integrin receptor was opposite that for {alpha}7ß1, in that the {alpha}6ß1 receptor was expressed strongly in the highly metastatic tumorigenic cell lines M-2, M-4, and C-26 and poorly in the nonmetastatic cells (25 , 26) . Cells that retained expression of {alpha}7 (for example, C-23, C-19, and C-10) or had forced expression of {alpha}7 (for example, M-2-{alpha}7), were less tumorigenic and less motile and metastasized infrequently (25 , 26) . Northern blot analysis demonstrated a good correlation between the level of {alpha}6 and {alpha}7 protein and the corresponding RNA transcripts. It is possible that high levels of {alpha}7 expression may directly regulate the levels of {alpha}6. However, indirect mechanisms could also account for the reduction in {alpha}6 expression when {alpha}7 is present. Finally, the parental K1735 cell line, which was established from a primary tumor, is composed of a mixture of phenotypically different clones with metastatic heterogeneity (25 , 26 , 30 , 33) . It is not surprising that the integrin profile of the parental cell line represents a composite of that displayed by the metastatic and nonmetastatic cell types. In conclusion, our results suggest that during tumor progression, there is selection of cells with an invasive phenotype that includes an integrin repertoire enriched in specific receptors that bind laminin transiently (e.g., {alpha}6) but lacking others that appear to bind laminin more efficiently (e.g., {alpha}7). Further work is required to determine the factors regulating {alpha}7 expression and how, once expressed, this laminin-binding receptor governs the growth and migratory properties of melanoma.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture and Materials.
The murine melanoma K1735 highly metastatic and low to nonmetastatic clones were obtained from Dr. I. J. Fidler (M. D. Anderson Cancer Center, Houston, TX). Cells were maintained in DMEM H16 with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and penicillin/streptomycin. Laminin-1 was purified from mouse Engelbreth-Holm-Swarm tumor as described previously (36) . Type I collagen was obtained from Collagen Biomaterials (Palo Alto, CA).

Antibodies against integrin subunits included mouse monoclonal antibodies to {alpha}1ß1 (Ha 31/8), ß1 (Ha 2/11; Ref. 61 ), {alpha}5ß1 (CD49E; PharMingen, San Diego, CA), {alpha}6ß1 (GoH3; PharMingen), ß1 (AIIB2; kindly provided by Dr. C. Damsky, University of California San Francisco), and {alpha}7ß1 (CY8; Ref. 20 ). The rabbit polyclonal anti-{alpha}7 antibody 1211 was prepared in this laboratory as described previously (62) , and the polyclonal anti-laminin-E8 antibody was a kind gift from Dr. P. D. Yurchenco (63) . Goat antirabbit IgG conjugated to HRP and the ECL kit were purchased from Amersham (Arlington Heights, IL).

Specific pathogen-free 6–8-week-old inbred C3H/HeN and athymic Balb/C mice were obtained from the Simonsen Laboratories, Inc. (Gilroy, CA). Mice were housed under specific pathogen-free conditions.

Cell Adhesion Assay.
Microtiter plates (96-well Immulon plates; Dynatech) were coated with ECM proteins at the indicated concentrations in PBS for 1 h at 37°C in a humidified atmosphere. Plates were washed with PBS and incubated with medium containing 0.1% BSA for 60 min in a CO2 incubator to block nonspecific adhesion. Single-cell suspensions were prepared in DMEM with 0.1% BSA at 4 x 105 cells/ml, added in triplicate to 96-well plates, and then incubated for 30–60 min at 37°C. Nonadherent cells were removed by shaking on a titer plate shaker and washed with PBS. Cells were fixed with 1% formaldehyde, stained with 1% crystal violet, and solubilized in 2% SDS; absorbance was then read at 562 nm. Binding of cells to collagen (10 µg/ml) on a separate plate was used to represent 100% attachment. Background cell adhesion to 1% BSA-coated wells was subtracted from all readings. The effect of specific blocking antibodies was tested by preincubating the cells with the indicated dilutions of purified antibodies on ice for 30 min prior to the assay.

Migration Assay.
Cell migration on laminin-1-coated surfaces was measured using the microscreen assay (64) . Briefly, sheets of virgin polystyrene were assembled in a 96-well dot blot apparatus (Schleicher and Schuell, Keene, NH); the wells were coated with laminin-1 for 1 h at the indicated concentrations. After washing, the apparatus was reassembled with the laminin-1-coated sheet and a polished stainless steel screen containing 0.9-mm-diameter perforations. Cells were seeded onto the exposed surface of the polystyrene sheet (1 x 105/ml per well). After a 1-h incubation at 37°C to permit cell attachment, the screen was removed, the apparatus was reassembled, and the cells were incubated for 8 h. Cells on the sheet were then processed by fixation with 0.5% formaldehyde and stained with hematoxylin. The area covered by out-migrating cells, from the fixed diameter of the microscreen, was measured by computer-assisted image analysis (NIH Image); the data were expressed as the mean and SD of at least five individual measurements in pixel units (x 103) and represent "relative migration."

Western Blot.
Retrovirally infected cell lines, parental cells, and tumors derived from each were solubilized with SDS-solubilization buffer [50 mM Tris (pH 7.5), 0.5% Triton X-100, 1 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide]. Equal amounts of protein were separated by SDS-PAGE on 7.5% polyacrylamide gels under nonreducing and reducing conditions (using 2-mercaptoethanol as reducing agent), transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and incubated with anti-{alpha}7 polyclonal antibody 1211 followed by goat antirabbit IgG-HRP or IgG-AP. Migration of the {alpha}7 subunit was determined, where indicated, by using an ECL detection system for HRP (Amersham) or the color development detection system for AP (Promega, Madison, WI).

Flow Cytometry.
After detachment with a brief treatment with 0.25% trypsin, single-cell suspensions of 106 cells/ml were incubated with optimal concentrations of primary antibodies in wash buffer (2% normal goat serum in PBS) for 1 h on ice. Cells were washed three times and incubated with secondary fluorescein-labeled antibodies for 30 min on ice. After three more washes, the cells were stained with propidium iodide (1 µg/ml) to identify nonviable cells. Flow cytometry was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Control samples consisted of cells with or without secondary antibody binding. Nonviable cells stained with propidium iodide were eliminated from the analysis. The cell populations, Mock and M-2-{alpha}7, were obtained by FACS sorting.

RNA Isolation and Northern Blot.
Total RNA was isolated by the guanidium isothiocyanate/phenol method and analyzed by Northern blot, as described previously (65) . Briefly, RNA samples were electrophoresed in a 1.2% agarose gel containing formaldehyde. Then the RNA was transferred to nylon membranes by capillary blotting and fixed to the filter by exposure to UV light. The RNA was hybridized with an {alpha}6 or an {alpha}7 cDNA fragment labeled with 32P. Hybridizations were carried out at 42°C in 50% formamide, 5x SSC, 5x Denhardt’s solution, 0.1% SDS, and 300 µg/ml salmon sperm DNA. Filters were washed twice in 1x SSC-0.1% SDS at room temperature and once at 65°C in 0.1x SCC-0.1% SDS. Filters were exposed to X-ray film at -80°C with intensifying screens.

Transfection.
The construction of {alpha}7-X2B cDNA has been described previously (62) . The murine {alpha}7-X2B cDNA was ligated into the retroviral expression vector pLNCX-4 (32) . Retroviral particles were obtained after transient transfection of the ecotropic packing cell line {psi}2 (obtained from the American Type Culture Collection, Manassas, VA). For transduction, culture medium was removed from M-2 cells and replaced with viral supernatant containing 8 µg/ml polybrene. After overnight incubation, the viral supernatant was removed and replaced with culture medium containing 800 µg/ml G418. After selection with G418, the retrovirally transfected cell populations M-2-{alpha}7 and Mock were FACS-sorted for {alpha}7 expression and lack of {alpha}7 expression, respectively. Expression of {alpha}7 was verified by Western blot analysis using polyclonal antibody 1211 (62) .

Tumorigenicity Studies.
Subconfluent cultures of M-2, Mock, and M-2-{alpha}7 cells were harvested by a brief incubation with a solution of 0.25% trypsin and 0.02% EDTA. Cells were washed in M-2 medium containing 10% FBS, centrifuged at 4°C, and resuspended in Ca2+- and Mg 2+-free HBSS at a concentration of 1 x 106 cells/ml. Only single-cell suspensions of >90% viability, as determined by trypan blue exclusion, were used. Groups of six C3H/HeN and six athymic mice were given s.c. injections containing 4 x 105 tumor cells. The growth rate of the s.c. tumors was monitored at the indicated times by examination of the mice and measurement of the tumors with calipers.

Experimental Metastasis.
Unanesthetized syngeneic C3H/HeN mice were given injections in the lateral tail vein with Ca2+- and Mg 2+-free HBSS containing 1 x 105 tumor cells. Mice were sacrificed 2 weeks after tumor cell injection and necropsied. The lungs were removed, rinsed in distilled water, and fixed in Bouin’s solution (25) . The number of surface tumor nodules was determined with the aid of a dissecting microscope.

In Vitro Growth Assays.
The growth rates of M-2 and M-2-{alpha}7 cells in medium supplemented with high and low concentrations of serum were compared to determine whether serum dependence was altered upon transfection of the {alpha}7 subunit. M-2 and M-2-{alpha}7 cells (104) were plated in 60-mm tissue culture dishes in 3 ml of medium containing 10% FBS. After incubation for 24 h, the medium was aspirated, and the monolayers were washed twice with serum-free DMEM H16 and then refed with DMEM H16 supplemented with either 10 or 0.5% FBS. After 5 days of incubation at 37°C, proliferation rates from triplicate dishes were determined.

Proliferation rates of M-2 and M-2-{alpha}7 cells were also compared to determine whether transfection of the {alpha}7 subunit affected the growth of these cells when plated on plastic versus laminin-1. For growth on laminin-1, microtiter plates (96-well Immulon plates) were precoated with 10 µg/ml of laminin-1 in PBS for 1 h at 37°C in a humidified atmosphere. For growth on plastic, tissue culture-treated microtiter plates were used. Cell lines were plated at a density of 50 cells per well. On the indicated days, cell numbers from triplicate wells were determined.

Changes in proliferation of M-2 and M-2-{alpha}7 cells under the conditions above were detected by using the CellTiter 96 AQueous one-solution cell proliferation assay according to the manufacturer’s instructions (Promega).


    Acknowledgments
 
We thank Dr. Lucia Belviglia for assistance with the tumorigenicity and metastasis studies and Evangeline Leash for editorial assistance.


    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 This work was supported by NIH Grants DE 13479 and DE 11912 and by a University of California Individual Investigator Research Award (to B. L. Z.). Back

2 Present address: Department of Otorhinolaryngology, University of Pennsylvania, Philadelphia, PA 10104. Back

3 To whom requests for reprints should be addressed, at, Department of Stomatology, University of California San Francisco, San Francisco, CA 94143-0512. Phone: (415) 476-3274; Fax: (415) 476-4204; E-mail: randyk{at}itsa.ucsf.edu Back

4 The abbreviations used are: ECM, extracellular matrix; FACS, fluorescence-activated cell sorting; HRP, horseradish peroxidase; AP, alkaline phosphatase. Back

Received for publication 1/25/99. Revision received 5/24/99. Accepted for publication 5/27/99.


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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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