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[Cancer Research 59, 1115-1119, March 1, 1999]
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[Cancer Research 59, 1115-1119, March 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Use of A Novel Fibronectin Receptor for Liver Infiltration by a Mouse Lymphoma Cell Line RL-1

Mozammel Haque Gazi and Masafumi Ito1

Department of Pathology, Yamagata University School of Medicine, Yamagata 990-9585, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism whereby some lymphomas invade liver extensively has not been fully investigated. There is no basement membrane under the sinusoidal endothelium of the liver, and hepatocytes produce fibronectin (FN); therefore, adhesion to this FN may be particularly important for liver infiltration by lymphoma cells. A mouse lymphoma cell line, RL-1, adhered to FN. However, this cell line did not express classical FN receptors such as very late antigen (VLA)-4 and VLA-5, as estimated by immunofluorescent staining. We have generated monoclonal antibodies (mAbs) that inhibit adhesion of RL-1 cells to FN. Western blot and immunoprecipitation analyses showed that the new mAbs recognize a protein with an approximate molecular weight of 55,000 (p55). This antigenic protein was highly purified by immunoprecipitation and processed for microsequencing. From NH2-terminal sequence results, the p55 antigen was not identical to known FN receptors. Radioisotope-labeled RL-1 cells, when injected i.v. into mice, rapidly infiltrated the liver (30–35% of injected cells), as measured by a gamma counter. Intravenous injection of the new mAbs partially (20%) blocked the infiltration of i.v.-injected lymphoma cells into the liver, whereas control rat IgG and an anti-CD11a mAb did not. These results demonstrate that the mouse lymphoma cell line RL-1 uses a novel FN receptor for liver infiltration.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For many tumor cell types, the liver is a major site of metastasis. Some lymphomas invade liver tissue and spread diffusely without forming nodules (1, 2, 3) . The blood-borne tumor cells are arrested in the sinusoidal endothelium of the liver. Malignant cells that metastasize from the primary site to other tissues may use the same adhesion molecules used by normal cells for traffic and localization in various organs and inflamed tissues.

LFA-12 is a receptor for intercellular adhesion molecule-1, and the LFA/ICAM-1 pathway plays an important role in the regulation of a variety of immune responses (4) . Several studies suggest that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells (5, 6, 7) . However, little is known about another pair of adhesion molecules, FN and its receptors. Although most lymphomas do not express known FN receptors such as VLA-4 and VLA-5, adhesion to FN, which is present in abundance on the hepatocyte surface (8) , may be important for metastasis to the liver because there is no basement membrane under the sinusoidal endothelium; therefore, tumor cells cannot adhere to laminin and collagen type IV as in other organs.

To address this possibility, we tried to make a mAb that inhibits binding to FN of a mouse lymphoma cell line, RL-1, which has no VLA-{alpha}4, VLA-{alpha}5, or VLA-ß1. We report the molecular characterization of a novel FN receptor that is recognized by our mAbs and the in vivo effect of these mAbs on liver infiltration by RL-1 lymphoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
BALB/c mice and BALB/c nude mice, 6–8 weeks of age, were purchased from Japan SLC, Inc. (Hamamatsu, Japan). F344 rats, 6 weeks of age, were purchased from Japan Charles River Company (Atsugi, Japan). The animals were kept in the facilities at Animal Research Laboratory of Yamagata University School of Medicine.

Cells and Cell Culture.
The mouse lymphoma cell line RL-1 was obtained from National Cancer Institute (Bethesda, MD) and maintained in our laboratory (9) . The human erythroleukemia cell line K562 was obtained from American Type Culture Collection (Rockville, MD). All cell lines were maintained in RPMI 1640 (Nikken Bio Medical Laboratory, Kyoto, Japan), supplemented with 10% fetal bovine serum (Biocell Laboratories, Inc., Rancho Dominguez, CA) and 50 µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO). Splenic T lymphocytes were isolated by mincing the spleen, lysing the red cells by ammonium chloride, and passing the cells through a nylon wool column.

Antibodies.
Rat mAbs specific for Thyl.2 (30-H12), LFA-1 (M17/4), VLA-4 {alpha} chain (9C10), VLA-5 {alpha} chain (5H10-27), and integrin-ß1 chain (9EG7) were purchased from PharMingen (San Diego, CA). The mouse mAb directed against the cell binding site of human plasma FN (790D24) was purchased from Chemicon International, Inc. (Temecula, CA). FITC-conjugated, peroxidase-conjugated, alkaline phosphatase-conjugated, and biotin-conjugated F(ab')2 fragments of mouse anti-rat IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA).

Generation of mAbs.
Female F344 rats were immunized i.p. with 107 RL-1 cells in PBS four times at 7-day intervals. Hybridoma production was done essentially as described (10) , except that the mouse myeloma Sp2/0 was used for the fusion. Hybridoma supernatants were screened by a cell adhesion inhibition assay (see below). The mAbs LAD-4 (IgG2b/{kappa}) and H-4 (IgG2a/{kappa}) were purified from ascites produced in pristane-primed BALB/c nude mice and from culture supernatants on a protein G-Sepharose column (Pharmacia, Uppsala, Sweden).

Cell Adhesion Assay.
Cell adhesion to plate-bound ECM proteins was assayed as described elsewhere (11) , with some modification. ECM used are human FN (Sigma), human collagen type IV (Sigma), and human laminin (Sigma). We used 1% skim milk (Difco Laboratories, Detroit, MI) for blocking nonspecific binding. Anti-FN mAb (10 µg/ml) and peptides (50 µg/ml) such as Arg-Gly-Asp-Ser (RGDS), Arg-Gly-Glu-Ser (RGES), and CS-1 (Peptide Institute, Osaka, Japan) were added to the cells before plating. In some experiments, cells were stimulated with 10 ng/ml PMA and 1 µM ionomycin for 10 min at 37°C before plating. The bound cells were measured by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (Chemicon).

Immunofluorescence.
Cells were incubated with the indicated mAb for 30 min on ice as described previously (12) . After incubation of FITC-conjugated mouse anti-Rat IgG, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickenson, Mountain View, CA).

Western Blot.
RL-1 cells were extracted in a lysis buffer (1% Triton X-100, 50 mM Tris-HCL, 150 mM NaCl, 2 mM CaCl2, and 10 µM phenylmethylsulfonyl fluoride). Proteins were separated by SDS-PAGE on 10% gels (ATTO Co., Tokyo, Japan) and were electroblotted onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA) as described previously (13) . The blots were incubated with the indicated rat mAbs, followed by peroxidase-conjugated mouse anti-rat IgG. The blots were subsequently incubated with Vectasain ABC solution (Vector Laboratories, Inc., Burlingame, CA). After being washed, the blots were further incubated with chemiluminesence reagents (New England Nuclear, Boston, MA) and exposed to X-ray films for 30 s to 1 min.

Cell Surface Biotinylation and Immunoprecipitation.
RL-1 cells (1 x 107) were labeled with 100 µg/ml of sulfo-N-hydrosuccinimide biotin (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. The biotin-labeled cells were extracted in a lysis buffer (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 3 mM EDTA, 1 mM PMSF, and 0.1% azide). After preclearing with normal rat IgG adsorbed protein G-Sepharose, the lysates were incubated with 50 µg of LAD-4 mAb or a control mAb (anti-Thy1.2, IgG2b) for 1 h on ice, followed by 10 µl of protein G-Sepharose beads for 30 min on ice. After washing with the lysis buffer, antigens were eluted from the beads with 40 µl of Laemmli’s sample buffer, subjected to SDS-PAGE, and electroblotted onto nitrocellulose membrane filters (Nihon Millipore Ltd., Tokyo, Japan). The blots were incubated with Vectasain ABC solution, followed by chemiluminescence reagents, and exposed to X-ray films for 3–5 min as described above.

Purification and Sequencing of the LAD-4 Antigen.
RL-1 cells (1 x 108) were immunoprecipitated with 100 µg of LAD-4 mAb as described above. After being washed three times in a buffer (0.5 M NaCl, 0.1% SDS, 0.01% Tween 20, and 50 mM Tris-HCl) followed by a wash in a buffer without NaCl, the immunoprecipitate was eluted from the protein G-Sepharose beads with 50 µl of sample buffer, and subjected to SDS-PAGE, and electroblotted onto PVDF membranes. Two lanes of the PVDF membranes were stained with Coomassie Blue and destained as described (14) . Because protein bands were undetectable with Coomassie Blue dye, proteins in the adjacent lanes were probed with LAD-4 mAb, followed by alkaline phosphatase-conjugated mouse anti-rat IgG, for use as markers to facilitate localization of the antigen. Color was developed using ß-naphthyl phosphate and Fast Blue BB salt (Sigma) as described (13) . Some were stained by colloidal gold (Bio-Rad) to assess yield and purity. The p55-containing parts of the PVDF membrane were excised, and at least eight membrane pieces were stored in a microfuge tube at -20°C until microsequencing was performed using the Hewlett-Packard G1005A protein sequence analysis system at Center for Research and Education in our university.

In Vivo Cell Infiltration Assay.
RL-1 cells were radiolabeled with 3.7–7.4 MBq of Na251 CrO4 (New England Nuclear) as described (12) and resuspended in PBS containing the mAb LAD-4, the mAb H-4, the anti-LFA-1 mAb (M17/4), or control rat IgG. Mice (five mice/group) received i.v. injections of 0.1 ml of suspension that contained 100 µg of each mAb and 1 x 106 RL-1 cells labeled with 1 x 106 cpm of 51Cr. After 24 h, the liver, spleen, and lungs were removed, and their radioisotope content was determined with a gamma counter. In some experiments, mice received i.v. injections of 51Cr-labeled RL-1 cells in the absence of antibody, and then after 1 h, the radioisotope content of each organ was determined.

Statistical Analysis.
Differences between the results of experimental treatments were evaluated by means of one factor ANOVA with Scheffe’s (posthoc) test. All values are expressed as means ± SD, and statistical significance was set at P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adhesion of Mouse Lymphoma Cell Line RL-1 to ECM Proteins.
We first examined the adherence of a mouse lymphoma cell line, RL-1, to various ECM proteins coated onto plates. RL-1 cells had a weak binding capacity for FN but did not bind to collagen type IV and laminin. Binding of RL-1 cells to FN was strongly enhanced by stimulation with PMA (Fig. 1)Citation .



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  		Fig. 1. Adhesion of RL-1 cells to ECM proteins in the presence or absence of PMA. Data represent means of triplicate samples; bars, SD. LM, laminin; CL, collagen type IV; BSA, bovine serum albumin.

 
Lack of Expression of VLA-4 and VLA-5 on RL-1 Cells.
Because RL-1 cell binding to FN suggested the expression of known FN receptors such as VLA-4 and VLA-5 (15) , we tested for the expression of these antigens by flow cytometry. RL-1 cells did not express VLA-{alpha}4, VLA-{alpha}5, or VLA-ß1, whereas they did express Thy1.2 and LFA-1 (CD11a; Fig. 2Citation ).



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  		Fig. 2. Lack of expression of VLA-4 ({alpha}4), VLA-5 ({alpha}5), and VLA-ß1 on RL-1 cells. Solid lines, staining with the first layer mAb: anti-Thy1.2, anti-LFA-1, the new mAb LAD-4, anti-VLA-{alpha}4, anti-VLA-{alpha}5, and anti-VLA-ß1. Dotted lines, staining with control rat IgG.

 
Inhibition of RL-1 Cell Binding to FN by New mAbs.
In spite of lacking known FN receptors, RL-1 cells bound strongly to FN. This suggested the expression of a novel FN receptor by RL-1 cells. To generate a mAb that inhibits binding of RL-1 cells to FN, we immunized rats with RL-1 cells, and the resulting immune splenocytes were fused with Sp2/0 myeloma cells. After screening the hybridoma culture supernatants for the ability to inhibit binding of RL-1 cells to FN, LAD-4 and H-4 were found to inhibit the adherence of the cells to FN (Fig. 3B)Citation . None of the mAbs that recognized VLA-{alpha}4, VLA-{alpha}5, or VLA-ß1 had an inhibitory effect (Fig. 3B)Citation , in contrast to the positive controls, an anti-FN mAb, and Arg-Gly-Asp-Ser (RGDS) peptides (Fig. 3A)Citation . These results show that RL-1 cells bind to the RGD site of FN.



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  		Fig. 3. Effect of various mAbs and FN-related peptides on RL-1 cell binding to FN. In A, RL-1 cells stimulated with PMA were added to FN-coated plates and incubated in the presence of RGD, CS-1 peptides, or anti-FN mAb for 1 h. In B, RL-1 cells stimulated with PMA were preincubated with each mAb for 30 min. The cells were then added to FN-coated plates and incubated for 1 h. The results are indicated as percentage of adherence, compared with the absorbance values of total RL-1 cells. Data represent means of triplicate samples; bars, SD.

 
Expression of the Antigen Recognized by the New mAbs on Various Cells.
Fluorocytometry with a new mAb, H-4, revealed that both RL-1 cells and splenic T cells, but not K562 cells, expressed the novel FN receptor (Fig. 4)Citation .



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  		Fig. 4. Expression of the antigen recognized by the new mAb on various cells. The type of stained cells (RL-1, splenic T, or K562) are indicated in each panel. Solid lines, staining with the new mAb H-4. Dotted lines, staining with control rat IgG.

 
Molecular Characterization of the Antigen Recognized by the New mAbs.
In Western blots of whole-cell lysates of RL-1 cells, both mAbs LAD-4 and H-4 identified the same band of a protein with an approximate Mr of 55,000–60,000 in nonreducing conditions (Fig. 5ACitation , Lane 5, and Fig. 5BCitation , Lane 4), whereas the anti-Thy1.2 mAb identified a band migrating at an approximate Mr 24,000 (Fig. 5A, Lane 2)Citation . In addition, our mAb did not react with proteins on a Western blot of K562 cell lysates (Fig. 5A, Lane 3)Citation . The Mr 55,000–60,000 was also detected when normal spleen cells were subjected to a similar analysis (Fig. 5B, Lane 3)Citation . Under reducing conditions, no band was detected by these mAbs (Fig. 5A, Lanes 1 and 4)Citation , suggesting that our mAbs recognize a conformation-specific epitope. The anti-integrin-ß1 mAb detected a Mr 110,000 protein on a Western blot of spleen cell lysates but not of RL-1 cell lysates (Fig. 5B)Citation , thus confirming the data obtained with immunofluorescent staining.



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  		Fig. 5. Molecular characterization of the antigen recognized by the new mAbs. In A, whole-cell lysates of RL-1 (Lanes 1, 2, 4, and 5) and K562 (Lane 3) were probed with anti-Thy1.2 (Lanes 1 and 2) and the mAb LAD-4 (Lanes 3–5) on Western blot. Right, position of Mr standards. x1000. In B, whole-cell lysates of spleen cells (Lanes 1 and 3) and RL-1 cells (Lanes 2 and 4) were probed with anti-VLA-ß1 mAb (Lanes 1 and 2) and the new mAb H-4 (Lanes 3 and 4) on a Western blot. In C, the antigens were immunoprecipitated from surface biotinylated RL-1 cell lysates with mAb LAD-4 (Lanes 2 and 3) or anti-Thy1.2 mAb (Lanes 1 and 4).

 
To further characterize the protein recognized by the mAb LAD-4, we performed immunoprecipitation analysis. In the presence of a chelating agent, EDTA, LAD-4 precipitated a protein (p55) with an approximate Mr of 55,000 under nonreducing conditions from a surface-biotinylated RL-1 cell lysate (Fig. 5C)Citation , indicating that this mAb recognized a 55,000 polypeptide on the surface of the RL-1 cells.

To determine the identity of the antigen recognized by LAD-4, the antigenic protein was highly purified by immunoprecipitation. After extensive washing in a buffer containing 0.5 M NaCl and 0.1% SDS, the immunoprecipitate was subjected to SDS-PAGE and electroblotted onto a PVDF membrane. The p55-containing parts of the membrane, although undetectable with Coomassie Blue dye, were excised by comparison with the markers in the adjacent lanes and processed for microsequencing. The sequences of the first 16 NH2-terminal amino acids were obtained (Fig. 6A)Citation and compared with those of known FN receptors (Fig. 6B)Citation (16, 17, 18, 19, 20, 21) . The p55 sequence was quite different from other integrin subunit sequences at positions 1–6, where VLA {alpha} subunits show striking homology (Fig. 6)Citation .



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  		Fig. 6. Comparison of NH2-terminal amino acid sequences from p55 with VLA protein subunits. In A, the LAD-4 antigen (p55) was purified by immunoprecipitation from RL-1 cell lysates and processed for microsequencing. The identical NH2-terminal amino acid sequence was obtained. In B, NH2-terminal sequences for mouse, m{alpha}3 (16) , m{alpha}4 (17) , and mß1, (18) and for human, h{alpha}3 (19) , h{alpha}4 (20) , and hß1 (21) , are translated from cDNA data.

 
Effect of the New mAbs on Liver Infiltration by RL-1 Lymphoma Cells.
Our molecular analysis showed that RL-1 cells express a novel FN receptor, the molecular mass of which differs from those of VLA proteins (15) . To test whether RL-1 cells use this FN receptor in the infiltration process and to determine the ability of our mAbs to block the infiltration of RL-1 cells into the liver, we undertook the following experimental protocol.

In the first experiment, BALB/C mice were injected i.v. with 1 x 106 51Cr-labeled RL-1 cells. After 1 and 24 h, the mice were killed, and their organs were removed. The percentage of infiltration in each organ was calculated from the radioisotope count (cpm) of each organ divided by the total cpm of 1 x 106 RL-1 cells. After 1 h, 20% of the injected cells had accumulated in the lungs, 28% in the liver, and <1% in the other organs (Fig. 7A)Citation . However, after 24 h, 35% of the injected cells still remained in the liver, whereas <1% were present in the lungs as well as the other organs (Fig. 7A)Citation , showing that RL-1 cells have an affinity for the liver.



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  		Fig. 7. Effect of new mAbs on liver infiltration by RL-1 lymphoma cells. In A, BALB/c mice (five mice/group) received i.v. injections of 106 51Cr-labeled RL-1 cells. After 1 and 24 h, each organ (liver, spleen, and lungs) was removed, and their radioisotope contents were determined. The results were indicated as percentage of infiltration, compared with the total counts (cpm) of RL-1 cells; bars, SD. In B, BALB/c mice received i.v. injections of RL-1 cells and 100 µg of each mAb. After 24 h, the percentage of infiltration in each organ was determined as in A. *, P < 0.05 by one factor ANOVA with Scheffe’s (posthoc) test; bars, SD.

 
In the next experiment, mice received i.v. injections of 1 x 106 51Cr-labeled RL-1 cells and 100 µg of our mAb (LAD-4 or H-4) or a control mAb such as normal rat IgG and an anti-LFA-1 mAb (M17/4). After 24 h, the percentage of infiltration in each organ was determined. Our mAbs, but not the other Abs, inhibited RL-1 cell infiltration into the liver by 20% (P < 0.05; Fig. 7BCitation ). The extent of the inhibition was not increased by injection of 500 µg of the mAb LAD-4 (data not shown). Because the anti-LFA-1 mAb was derived from the same species (rat) and was of the same isotype (IgG2a) as the mAb H-4, the inhibitory effect of our mAbs on liver infiltration seems to be specific.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor cells can use adhesion molecules both for crossing endothelial cells and for settling in target organs. Both the malignant and normal cells may use the same mechanism of traffic and localization. For instance, LFA-1 is implicated in inflammatory and immune responses via cell-cell and, to a lesser extent, cell-matrix interactions (22, 23, 24) . Modulation of LFA-1 expression has been shown to be associated with the invasion of mouse lymphoma cell lines into hepatocyte cultures (5) and in lymphoma metastasis (6) . Some antibodies directed against LFA-1 inhibit invasion by murine and human lymphoma cell lines (7) .

By contrast, the role of FN receptors on lymphoma cells in metastasis to the liver is largely unknown, although several studies have shown that RGD peptides, a minimal crucial sequence for cell attachment (24 , 25) , inhibit melanoma invasion of the lung (26, 27, 28) , and that both RGD peptides and anti-FN polyclonal antibodies block adhesion of murine mammary carcinoma cells to hepatocytes in vitro (29) . Because hepatocytes synthesize FN and carry it on their surface, a mAb directed against the FN receptor may inhibit lymphoma spreading in the liver if the lymphoma cells express the FN receptor. Our findings show that RL-1 lymphoma cells express a novel FN receptor consisting of a Mr 55,000–60,000 protein but that they lack classical FN receptors such as VLA-4 and VLA-5. Moreover, this receptor differs from other adhesion receptors for FN such as VLA-3 and vitronectin receptor (15 , 24) in terms of molecular size and NH2-terminal amino acid sequence. Although our sequencing data may not be complete, the sequences at positions 1–6 were reproducible, and a difference at these positions between p55 antigen and other VLA subunits was evident, indicating that p55 antigen is a novel FN receptor. To further investigate the homologies between p55 antigen and VLA protein subunits, the complete cDNA sequence of p55 antigen must be obtained.

Our newly established mAbs injected i.v. reduced the infiltration of RL-1 lymphoma cells into the liver. This effect on liver infiltration was partial. Therefore, the mechanism by which lymphoma cells infiltrate the liver may be complex, and several adhesion receptors can be involved.

It is interesting that an anti-LFA-1 mAb (M17/4) did not inhibit liver infiltration by RL-1 lymphoma cells. This is in agreement with Aoudjit et al. (30) , who demonstrated with ICAM-1-deficient mice that LFA-1/ICAM interaction is not critical for homing by lymphoma cells but is involved in liver metastasis, a posthoming event.

In conclusion, our study shows that some lymphoma cells can use a novel FN receptor to invade the liver, even if they lack classical FN receptors. Adhesion of lymphoma cells to hepatocyte FN may be the mechanism responsible for liver infiltration. However, it will be necessary to clarify the complete set of adhesion molecules present on tumor cells and hepatocytes to understand the role of adhesion in the formation of liver metastasis.


   ACKNOWLEDGMENTS
 
We thank Drs. Kan-etsu Sugawara and Seiji Hongo at the Department of Bacteriology for technical advice and critical comments and Drs. Suzuki and Sato at the Center for Research and Education of Yamagata University School of Medicine for protein sequencing. We also thank Dr. Yoichi Fujii at Nagoya University School of Medicine for critical comments and Dr. Shigeru Arai at Yamagata Prefectural Nihonkai Hospital for encouragement.


   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 To whom requests for reprints should be addressed, at Department of Pathology, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990-23, Japan. Back

2 The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1; ICAM-1, intercellular adhesion molecule-1; FN, fibronectin; VLA, very late activation antigen; mAb, monoclonal antibody; ECM, extracellular matrix; PVDF, polyvinylidene difluoride; PMA, phorbol myristate acetate. Back

Received 3/ 9/98. Accepted 1/ 4/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Roos E., Dingemans K. P., Van de Pavert I. V., Van den Bergh Weerman M. A. Invasion of lymphosarcoma cells into the perfused mouse liver. J. Natl. Cancer Inst., 58: 399-407, 1977.
  2. Rudders R., Levin A., Jespersen D., Zachs J., Delellis R., Ranger A., Kronitis T. Crossreacting human lymphoma idiotypes. Blood, 80: 1039-1044, 1992.[Abstract/Free Full Text]
  3. Krüger A., Schirrmacher V., von Hoegen P. Scattered micrometastasis visualized at the single-cell level: detection and re-isolation of lacZ-labeled metastasized lymphoma cells. Int. J. Cancer, 58: 275-284, 1994.[Medline]
  4. Springer T. A., Dustin M. L., Kishimoto T. K., Marlin S. D. The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Annu. Rev. Immunol., 5: 223-252, 1987.[Medline]
  5. Roosien F. F., Bikker A., De Rijk D., Roos E. Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants. J. Cell Biol., 108: 1979-1985, 1989.[Abstract/Free Full Text]
  6. Stauder R., Grei R., Schulz T. F., Gattringer C., Radaskiwicz T., Dierich M. P., Huber H. Expression of leukocyte function-associated antigen-1 and 7F7 antigen, an adhesion molecule related to intercellular adhesion molecules-1 (ICAM-1) in non-Hodgkin lymphomas and leukemias: possible influence of growth and leukaemic behaviour. Clin. Exp. Immunol., 77: 234-238, 1989.[Medline]
  7. Harning R., Myers C., Merluzzi V. J. Monoclonal antibodies to lymphocyte function-associated antigen-1 inhibit invasion of human lymphoma and metastasis of murine lymphoma. Clin. Exp. Metastasis, 11: 337-342, 1993.[Medline]
  8. Kemperman H., Driessens M. H. E., La Riviére G., Meijine A. M. L., Roos E. Adhesion mechanisms in liver metastasis formation. Cancer Surv., 24: 67-79, 1995.[Medline]
  9. Fuyama S., Sendo F., Watabe S., Seiji K., Arai S. Inhibition of mouse natural killer activity by cholera toxin. Gann, 72: 141-144, 1981.[Medline]
  10. Ito M., Suzuki E., Naiki M., Sendo F., Arai S. Carbohydrates as antigenic determinants of tumor-associated antigens recognized by monoclonal anti-tumor antibodies produced in a syngeneic system. Int. J. Cancer., 34: 689-697, 1984.[Medline]
  11. Miyake S., Sakurai T., Okumura K., Yagita H. Identification of collagen and laminin receptor integrin on murine T lymphocytes. Eur. J. Immunol., 24: 2000-2005, 1994.[Medline]
  12. Ito M., Fredrickson G. G., Celada F., Moran T. M., Bona C. A. Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor ß chain marker. J. Immunol. Methods, 103: 229-237, 1987.[Medline]
  13. Fujii Y., Ito M., Ikuta K. Evidence for the role of human immunodeficiency virus type 1 Nef protein as a growth inhibitor to CD4+ T lymphocytes and for the blocking of the Nef function by anti-Nef antibodies. Vaccine, 11: 837-847, 1993.[Medline]
  14. Matsudaira P. Sequences from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem., 262: 10035-10038, 1987.[Abstract/Free Full Text]
  15. Hemler M. E. VLA proteins in the integrin family: structures, function, and their role on leukocytes. Annu. Rev. Immunol., 8: 365-400, 1990.[Medline]
  16. Takeuchi K., Hirano K., Tsuji T., Osawa T., Irimura T. cDNA cloning of mouse VLA-3 subunit. J. Cell. Biochem., 57: 371-377, 1995.[Medline]
  17. Neuhaus H., Hu M. C., Hemler M. E., Takada Y., Holzmann B., Weissman I. L. Cloning and expression of cDNA for the alpha subunit of the murine lymphocyte-Peyer’s patch adhesion molecule. J. Cell Biol., 115: 1149-1158, 1991.[Abstract/Free Full Text]
  18. Holers V. M., Ruff T. G., Parks D. L., McDonald J. A., Ballard L. L., Brown E. J. Molecular cloning of a murine fibronectin receptor and its expression during inflammation. Expression of VLA-5 is increased in activated peritoneal macrophages in a manner discordant from major histocompatibility complex class II. J. Exp. Med., 169: 1589-1605, 1989.[Abstract/Free Full Text]
  19. Tsuji T., Hakomori S., Osawa T. Identification of human galactoprotein b3, an oncogenic transformation-induced membrane glycoprotein, as VLA-3 {alpha} subunit: the primary structure of human integrin a3. J. Biochem., 109: 659-665, 1991.[Abstract/Free Full Text]
  20. Takada Y., Elices M. J., Crouse C., Hemler M. E. The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function. EMBO J., 8: 1361-1368, 1989.[Medline]
  21. Argraves W. S., Suzuki S., Arai H., Thompson K., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the human fibronectin receptor. J. Cell Biol., 105: 1183-1190, 1987.[Abstract/Free Full Text]
  22. Springer T. A., Dustin M. L., Kishimoto T. K., Marlin S. D. The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Annu. Rev. Immunol., 5: 223-252, 1987.
  23. Oppenheimer-Marks N., Davis L. S., Lipsky P. E. Human T lymphocyte adhesion to endothelial cells and trans-endothelial migration: alteration of receptor use relates to the activation status of both the T cell and the endothelial cell. J. Immunol., 145: 140-148, 1990.[Abstract]
  24. Springer T. A. Adhesion receptors of the immune system. Nature (Lond.), 346: 425-433, 1990.[Medline]
  25. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science (Washington DC), 238: 491-497, 1987.[Abstract/Free Full Text]
  26. Humphries M. J., Olden K., Yamada K. M. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science (Washington DC), 233: 467-470, 1986.[Abstract/Free Full Text]
  27. Saiki I., Murata J., Iida J., Sakurai T., Nishi N., Matsuno K., Azuma I. Antimetastatic effects of synthetic polypeptides containing repeated structures of the cell adhesive Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR) sequences. Br. J. Cancer, 60: 722-728, 1989.[Medline]
  28. Ogawa R., Nishi N., Sugimura K., Tokura S., Azuma I. Inhibition of the metastasis of murine malignant melanoma by synthetic polymeric peptides containing core sequences of cell-adhesive molecules. Cancer Res., 49: 3815-3822, 1989.[Abstract/Free Full Text]
  29. Kemperman H., Wijnands Y., Meijne A. M. L., Roos E. TA3/St, but not TA3/Ha mammary carcinoma cell adhesion to hepatocytes is mediated by {alpha}5/ß1 interacting with surface associated fibronectin. Cell Adhesion Commun., 2: 45-48, 1994.[Medline]
  30. Aoudjit F., Potworowski E. F., Springer T. A., St-Pierre Y. Protection from lymphoma cell metastasis in ICAM-1 mutant mice: a posthoming event. J. Immunol., 161: 2333-2338, 1998.[Abstract/Free Full Text]




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