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Tumor Biology |
Metastasis Research Program, Karmanos Cancer Institute, Detroit, Michigan 48201 [H. C. G., Y. H., P. N-M., V. H., A. R.]; Department of Pathology, Radiation Oncology, Wayne State University, School of Medicine, Detroit, Michigan 48201 [A. R.]; Gastrointestinal Cancer Research Laboratory, Henry Ford Health Sciences Center, Detroit, Michigan 48202 [N. M., R. S. B.]
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ABSTRACT |
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-galactoside-binding protein family shown to be involved in tumor progression and metastasis. It has a unique primary structure consisting of three domains: a 12-amino acid leader sequence containing a casein kinase I serine phosphorylation site, which is preceded by a collagenase-sensitive Pro-Gly-rich motif, and a COOH-terminal half encompassing the carbohydrate-binding site. To study the functional role of the unusual leader sequence of galectin-3, a mutant cDNA that causes an 11-amino acid deletion in the NH2-terminal region was generated and expressed in galectin-3-null BT-549 human breast carcinoma cells. Deletion of the NH2 terminus resulted in abolition of the secretion of truncated galectin-3, loss of nuclear localization, and reduced carbohydrate-mediated functions compared with the wild-type protein. When green fluorescent protein was fused to the galectin-3 leader sequence and transiently transfected into BT-549 cells, the uniform cellular distribution of native green fluorescent protein was changed mainly to a nuclear pattern. To further investigate whether the functional changes observed in a galectin-3 with the 11 NH2-terminal amino acids deleted were due to loss of phosphorylation at Ser6, two point mutations were created at this serine: Ser6
Ala and Ser6Glu. No obvious difference was observed in cellular localization between wild-type and Ser6-mutated transfectants. These results suggest a structural role for the NH2 terminus leader motif of galectin-3 in determining its cellular targeting and biological functions independent of phosphorylation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Galectin-3 is found in the cytoplasm, on the cell surface, in the nucleus, and is secreted by tumor and inflammatory cells. Cell surface galectin-3 has been implicated in cellular recognition and adhesion during metastasis (7 , 8) , whereas nuclear galectin-3 has been associated with pre-mRNA splicing (9) . How galectin-3 is targeted to different cell compartments or secreted is not known because it contains no consensus leader sequence, but it has been suggested that the NH2 terminus is involved in some aspects of such targeting (10) . On the basis of the above and because the precise function of the 12-amino acid leader motif is obscure, we set out to test the hypothesis that this leader motif plays a role in cellular compartmentalization.
Galectin-3 has been shown to be phosphorylated at NH2-terminal Ser6 (major) and Ser12 (minor), and the acidic residues on both sides of Ser6 make this serine a likely substrate for casein kinase I and/or for casein kinase II (11) . The presence of both phosphorylated and nonphosphorylated galectin-3 has been reported in 3T3 fibroblasts; the phosphorylated form is found in both cytosolic and nuclear fractions, whereas the nonphosphorylated species is found exclusively in the nucleus (12) . Furthermore, cell proliferation is associated with an increased level of the phosphorylated species as well as nonphosphorylated derivatives (13) , and alterations in nuclear versus cytoplasmic galectin-3 localization have been shown to be associated with neoplastic progression (14 , 15) .
Galectin-3 is most likely synthesized on free cytoplasmic ribosomes. It lacks a typical signal sequence for translocation into the endoplasmic reticulum (16) and, therefore, does not follow the classical secretion pathway (16 , 17) . Secretion of galectin-3 is a relatively slow and incomplete process, taking >24 h for 20–30% of newly synthesized galectin-3 to be secreted, and the NH2-terminal domain has been implicated in this process (10 , 18) .
This study was designed to investigate the functional role of the 11 amino acids (following the first methionine) that constitute the NH2 terminus of galectin-3. Site-directed mutagenesis was used to generate and express a mutant cDNA encoding a galectin-3 protein that lacks the 11 NH2-terminal amino acids. Loss of the NH2 terminus resulted in alterations in galectin-3 cellular compartmentalization and function. To further address whether the loss of Ser6 phosphorylation by the action of casein kinase I led to changes of its biological function and cellular localization, two point mutations were generated at Ser6 (Ser6Ala and Ser6Glu). There was no obvious difference observed in cellular targeting of galectin-3 among wild-type and Ser6 point mutant transfectants.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Site-directed Mutagenesis.
To delete the NH2-terminal 11 amino acids (following the initiation codon), a sense primer (5'-CAGAATTCGGCTTATGGGGTCTGG-3') and an antisense primer (5'-CCAGACCCCATAAGCCGAATTCTG-3') containing a flanking EcoRI cleavage site were synthesized (Genosys, The Woodlands, TX). PCR-mediated site-directed mutagenesis was used to delete NH2-terminal amino acids 2 through 12, following the protocol supplied by the manufacturer (Stratagene, La Jolla, CA). PCR product was quantified and analyzed by gel electrophoresis. Approximately 10 ng of PCR product were ligated with pCRII vector (Invitrogen, San Diego, CA) at 14°C overnight. Two µl of the ligation reaction were used to transform One Shot competent cells (Promega, Madison, WI). A sense primer, 5'-CAATTTTGCGCTCCATGAT-3', and an antisense primer, 5'-ATCATGGAGCGCAAAATTG-3', for Ser6Ala point mutation, and a sense primer, 5'-ATGGCAGACAATTTTGACCT-3', and antisense primer, 5'-AGCTCAAAATTGTCTGCCAT-3', for Ser6Glu were ordered from IDT (Coralville, IA). Point mutations were introduced into pGEM(7+) vector using a QuickChange Site-Directed Mutagenesis Kit (La Jolla, CA). Briefly, 50 ng of pGEM(7+) vector containing wild-type galectin-3 cDNA were used as the template, two primers with Ser6 mutated were used for PCR to generate the point mutations. One µl of the DpnI restriction enzyme (10 units/µl) was added directly to each amplification reaction to cleave the template DNA. One µl of the DpnI-treated DNA was transferred into Epicurian Coli XL1-Blue supercompetent cells. Plasmid DNA was purified and sequenced. The sequence was confirmed by the Macromolecular Core Facility of Wayne State University.
Stable Transfection of Mutated Galectin-3 cDNA into Cell Line BT-549.
The mutant cDNA that codes for the deletion of 11 amino acids in the NH2-terminal region or for Ser6 point mutations in galectin-3 generated by site-directed mutagenesis was excised from the pCR II vector with EcoRI (Promega, Madison, WI) and inserted into the G418-resistant mammalian expression vectors pCNC10 and pBK-CMV, respectively, at the EcoRI site, generating the expression plasmids pCNC10-deletion of mutant galectin-3 and pBK-CMV-S6A and pBK-CMV-S6G of mutant galectin-3, with galectin-3 cDNA in the sense direction. The purified plasmid DNA of each mutant was used to transfect recipient BT-549 cells using Lipofectamine (Life Technologies, Gaithersburg, MD). G418-resistant cells were subcloned and four stable clones of the deletion mutant (Del 6, 7, 8, and 11) and two of each of serine point mutations (S6A 1 and 2 and S6G 1 and 2) were chosen for further analysis. Expression of galectin-3 in these clones and in the wild-type galectin-3 transfectant was detected by Western blot.
Western Blot Analysis of Wild-Type and Mutant Galectin-3 Expression.
Expression of galectin-3 was determined by immunoblotting. Briefly, 1 x 106 cells of wild-type galectin-3 transfectant cell line 11-9-14 (19)
, deletion mutant transfectant clones Del 6, 7, 8, and 11, and serine point mutation transfectant clones S6A 1, S6A 2, S6G 1, and S6G 2 were lysed with 50 µl of Triton X-100-containing lysis buffer (Fisher, Pittsburgh, PA). Protein concentrations were measured using bicinchoninic acid protein assay reagents (Pierce, Rockford, IL), and 50-µg aliquots of the cell lysates were separated by 12.5% SDS-PAGE and subjected to immunoblot analysis using both rat anti-galectin-3 antibody (mAb)3
TIB166 and rabbit anti-galectin-3 antiserum (pAb) as described previously (19)
. Duplicate gels were stained with Brilliant blue G-colloidal (Sigma, St. Louis, MO) to ensure equal loading. Molecular sizing was done using the Bench Mark Prestained Protein Ladder (Life Technologies).
Secretion of Galectin-3.
Cells (1 x 105) were grown in 60-mm plates until they were fully confluent, and then were washed twice with ice-cold CMF-PBS. One ml of serum-free medium was added to each plate, and the cells incubated at 37°C for 24 h. The medium was then collected and concentrated by centrifugation at 5000 rpm for 30 min with 10,000 NMWL UltraFree-MC filter unit (Millipore, Bedford, MA). The final volume was adjusted to 40 µl with PBS, and the secreted proteins were separated by 12.5% SDS-PAGE. Western blot analysis was used to determine the presence of galectin-3.
Immunofluorescence Labeling.
Cells were detached from 100-mm plates with 0.25% trypsin-EDTA (Life Technologies) and washed twice with PBS containing Ca2+ and Mg2+. Coverslips sterilized with methanol were placed into 6-well plates; 1 x 105 cells of 11-9-14 and Del 7 were seeded into each well, and the cells grown overnight in 10% DMEM. The cells were rinsed twice with PBS; for cell surface staining, they were fixed with 3.5% paraformaldehyde for 5 min on ice and 5 min at room temperature; for intracellular staining, cells were fixed and permeabilized with 100% methanol for 15 min at room temperature. Cells were then washed twice with PBS and blocked with 1% BSA in PBS. After a 30-min incubation on ice, rabbit anti-galectin-3 serum was added at a 1:50 dilution in 1% BSA and incubated 1 h at room temperature, followed by three washes with PBS. The following steps were performed in the dark. Secondary antibody (FITC goat antirabbit IgG; Zymed, San Francisco, CA; Ref. 19
) was added at a 1:200 dilution in 1% BSA and incubated 1 h, and then washed with PBS three times. The coverslips were transferred upside down onto glass slides with one drop of polyvinyl alcohol in PBS. Slides were then wrapped with aluminum foil and stored at 4°C until visualization.
Flow Cytometry.
Del 7 and 11-9-14 cells were harvested with 2 mM EDTA in CMF-PBS, washed three times with CMF-PBS, and resuspended at 1 x 106 cells/ml in ice-cold PBS containing 1% BSA. FACScan (Becton Dickinson, Bedford, MA) analysis was carried out as described previously (20
, 21)
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Hemagglutination Assay.
The hemagglutination assay was performed as described previously (22)
. Trypsin-treated, glutaraldehyde-fixed rabbit erythrocytes were prepared and stored at 4°C as a 10% stock in PBS containing 0.02% sodium azide. Prior to the assay, erythrocytes were diluted with 0.1 N glycine in PBS to yield a 4% suspension. Hemagglutination activity was measured by a series of 2-fold dilutions of cell extract with equal protein concentration in 96-well microtiter plates with V-shaped well bottoms. Each well contained 50 µl of cell extract diluted in CMF-PBS, 50 µl of 1% BSA in CMF-PBS, and 25 µl of a 4% erythrocyte suspension. The lowest dilution that caused distinct hemagglutination was determined after 1 h at room temperature.
Preparation of Nuclear Matrix Protein.
Nuclear matrices were prepared according to an established methodology (23)
. Cells were grown to confluence, detached with trypsin, centrifuged at 800 x g for 5 min, and resuspended in 400 µl of 0.5% ice-cold Triton X-100 in a buffered solution containing 2 mM RNase inhibitor (5prime3prime, Boulder, CO) to release the lipids and soluble proteins; the lysed cells were then precipitated at 16,000 rpm for 2 min. The pellets were washed twice to eliminate soluble proteins and were extracted with 0.25 M ammonium sulfate with RNase inhibitor to release the soluble cytoskeletal elements. This suspension was again spun at 16,000 rpm for 2 min. Ten µl of DNase I (Boehringer Mannheim, Indianapolis, IN) and 10 µl of RNase plus (5prime3prime, Boulder, CO) were added to the pellet with 100 µl of PBS, incubated at room temperature for 1 h, and then centrifuged as before to release the soluble chromatin and RNA. The pellets were then disassembled with 8 M urea, and the soluble components were separated by centrifugation. The protein concentration was determined, and 10 µg of nuclear proteins were separated on 12.5% SDS-PAGE, transferred to nitrocellulose filters, and probed for galectin-3 detection as described above.
Expression and Purification of S6A and S6G Mutant Proteins.
S6A and S6G mutant proteins were expressed in pBK-CMV expression vector and purified as described previously in detail (24)
. The lectin was dialyzed against 4 liters of 50 mM Tris buffer (pH 7.5).
Casein Kinase I Assay.
The casein kinase I-catalyzed incorporation of 32P into wild-type and S6A and S6G recombinant galectin-3 was assayed as described by Huflejt et al. (11)
. The reaction mixtures contained 50 mM Tris, 140 mM KCl, 10 mM MgCl2, 0.1 mM [
-32P]ATP, 39 microunits of purified casein kinase I (Calbiochem Corp., San Diego, CA), and 5 µg of each recombinant protein in a final volume of 50 µl. The reaction was carried out for 20 min at 30°C and was terminated by addition of 10 mM unlabeled ATP and 18 µl of 4x SDS-PAGE sample buffer. The samples were separated on 12% SDS-PAGE and visualized by autoradiography.
Anchorage-independent Growth and Tumorigenicity:
A single cell suspension of 1 x 103 cells of 11-9-14, Del 6, Del 7, Del 8, S6A 1, S6A 2, S6G 1, and S6G 2 in 1 ml of 0.5% agarose (Seaplaque; FMC, Rockland, ME) in DMEM (Life Technologies) plus 10% fetal bovine serum (Summit Biotech, Collins, CA) were placed in 35-mm plates on the top of 1% soft agarose that had been allowed to gel previously. The dishes were kept at 4°C for 2 h to solidify the cell-containing layer. The dishes were then incubated at 37°C in a CO2 incubator with 2 ml of medium. The medium was changed every 3 days. After 2 weeks, colonies larger than 10 cells were scored under a Nikon inverted-phase microscope. To determine in vivo tumorigenicity, 1 x 106 BT-549, 11-9-14, and Del 7 cells mixed with 2 ml of Matrigel were injected into the mammary fat pad of 6-to 8-week-old immune-deficient nude mice (NCR-NV; Taconic Farms, NY). Animals were assessed weekly for tumor development with calipers. Mice were sacrificed by CO2 inhalation 4 weeks after injection or when the tumors reached a size larger than 1.5 cm. The liver, lungs, and heart were removed and examined for metastases.
Construction of Fusion Protein.
Double-stranded cDNA that coded for the 11 NH2-terminal amino acids of galectin-3 with an EcoRI cleavage site at 5' and a BamHI cleavage site at 3' was generated by annealing two oligonucleotides—5'-AATTCCGCCACCATGGCAGACAATTTTTCGCTCCATGATGCGTTATCTG-3' and 5'-GATCCAGATAACGCATCATGGAGCGAAAAATTGTCTGCCATGGTGGCGG-3' (IDT, Coralville, IA)—using the following conditions: the temperature was raised to 94°C and slowly cooled to 25°C over 1 h. The same condition was use to generate double-stranded cDNA with Ser6 mutated to alanine and glutamic acid. pEGFP-N3 vector (Clontech, Palo Alto, CA) was digested with both EcoRI and BamHI and dephosphorylated with calf intestinal alkaline phosphatase. The double-stranded cDNA that coded for the 11 amino acids with or without serine mutations was ligated into pEGFP vector at room temperature for 2 h with T4 ligase. Two µl of ligation reaction were transformed into JM109 competent cells (Promega, Madison, WI). After 24 h, 10 colonies were chosen. Extracted DNA digested with EcoRI and BamHI showed both the 4.7-kb plasmid and a 50-bp insert. The DNAs were transfected into BT-549 cell using Lipofectamine (Life Technologies). The expressional distribution of GFP was examined under a fluorescence microscope after 2 h, according to manufacturer’s instructions (Clontech, Palo Alto, CA).
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RESULTS |
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Galectin-3 expression in BT-549 clones transfected with the galectin-3 deletion mutant was detected by the pAb but not by the mAb (Fig. 1)
, establishing the expression of mutant protein with the 11-amino acid deletion. Conversely, wild-type galectin-3 protein could be detected by both antibodies (Fig. 1)
. No galectin-3 was detected in vector-transfected control cells (Fig. 1)
. Among the four selected deletion mutant clones, Del 7 expressed the mutant galectin-3 at a level comparable to the wild-type galectin-3 transfectant cells and was thus chosen for subsequent studies. Similar levels of galectin-3 were also expressed by serine point mutation clones S6A 1 and S6G 1 (Fig. 2)
. The mutant transfectant cells were morphologically indistinguishable from parental BT-549 cells (Fig. 3, A, C, and D)
, but all differed from the wild-type transfectant cells, which showed a more rounded morphology without cytoplasmic protrusions (Fig. 3B)
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-32P]ATP after a 20-min incubation in 50 mM Tris buffer at 30°C. Wild-type galectin-3 was phosphorylated by casein kinase I, whereas no phosphorylation was detectable in S6A and S6G mutant proteins (Fig. 4)
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, whereas wild-type and Ser6 point mutant galectin-3 were detected in the conditioned media of the wild-type and mutant galectin-3-expressing cells (Fig. 5)
. Extracellular galectin-3 could not be detected in the concentrated conditioned media from the deletion mutants even after prolonged exposure of blots, suggesting that the galectin-3 NH2 terminus is involved in the secretory pathway, which does not depend on casein kinase I phosphorylation at Ser6.
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. Both galectin-3 species were detected in the cytoplasm of the respective cell clones. Nuclear localization of the wild-type galectin-3 was readily visualized (Fig. 6A)
in 11-9-14 cells, but no nuclear deposition could be detected in cells expressing the galectin-3 deletion mutant (Fig. 6B)
. To further validate the role of the NH2 terminus leader sequence of galectin-3 in regulating cellular translocation, nuclear matrix were extracted from cell cultures, separated by 12.5% SDS-PAGE, electrotransferred onto polyvinylidene fluoride membrane, and processed for immunodetection by anti-galectin-3 pAb. Galectin-3 could be detected both in the nuclear matrix of wild-type-transfected cells and Ser6Ala and Ser6Glu mutant clones (Fig. 7)
, whereas no nuclear-associated galectin-3 was detected in Del 7 cells; these results supported the immunofluorescence studies. In addition, expression of galectin-3 could be detected on the surface of wild-type galectin-3 transfectants (Fig. 6, C and D)
. The FAScan data showed the cell surface expression of galectin-3. We observed a shift of the fluorescence peaks in wild-type transfectants, which indicated surface expression of galectin-3. The peak on the left represents the negative control in the absence of primary antibody (Fig. 6C)
. Mutant clone Del 7 did not show the shift, which implies lack of mutant galectin expression on the cell surface (Fig. 6D)
. To further determine whether the leader sequence of galectin-3 is sufficient for directing cellular localization, we fused this sequence with or without Ser6 mutated with a GFP (pEGFP) vector. The leader sequences of galectin-3 was attached to the NH2 terminus of the pEGFP-N3-encoded protein, and the fused product and control unmodified GFP were transfected into BT-549 cells. Fig. 8A
shows that the intact GFP is uniformly distributed throughout the cytoplasm and nucleus, whereas the fluorescence of the fused proteins is predominantly localized in the nucleus (Fig. 8, B and C)
. This is similar to the effect of the PML proto-oncogene on the localization of fused-GFP to the nucleus (22)
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, whereas galectin-3 from deletion mutant clones Del 6 and 7 failed to promote hemagglutination. This suggests that the galectin-3 deletion mutant is present in the cell only as a monomer or has lost its carbohydrate-recognition ability. Recombinant galectin-3 from the deletion mutant was readily expressed by Escherichia coli but could not be purified on an asialofetuin affinity column like the wild-type species. This suggests that the mutated protein may be incorrectly folded, leading to a loss of the carbohydrate-binding ability.
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. There was no significant difference in the plating efficiencies of the three mutant transfectants in semi-solid medium compared with parental cell line BT-549, whereas plating efficiency of the wild-type transfectant 11-9-14 was significantly higher (Table 1)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fibroblast growth factor and interleukin 1 (10)
. It is believed that galectin-3 undergoes a post-translational modification after being synthesized on free cytoplasmic ribosomes and that the NH2-terminal Met and Ala could be acylated, thus blocking secretion and translocation of galectin-3 to the cell surface (25)
. Because wild-type galectin-3 is secreted into culture medium, whereas no galectin-3 secretion is observed in cells expressing the truncated protein, it is likely that the 11 NH2-terminal amino acids of galectin-3 are essential for secretion. Furthermore, the results suggest that inhibition of secretion is due to abrogation in galectin-3 translocation to the cell surface. It was documented previously that galectin-3 may undergo casein kinase I-catalyzed phosphorylation at Ser6 and Ser12 (major and minor sites, respectively; Ref. 11 ). The current study demonstrated that blocking of the phosphorylation of galectin-3 at Ser6 is not responsible for inhibition of secretion in NH2 terminus-deleted transfectant clones. It has also been postulated that charge alteration of this motif could affect galectin-3 interaction with its ligands (11) . The presence of both phosphorylated and nonphosphorylated endogenous galectin-3 has been reported. The phosphorylated form of galectin-3 was found in both cytosolic and nuclear fractions; whereas the nonphosphorylated species existed exclusively in the nucleus (12) . Furthermore, cell proliferation was associated with an increased level of the phosphorylated and nonphosphorylated galectin-3, which was manifested most clearly in the nuclear fraction (13) . If phosphorylation per se was the sole determinant of the cellular distribution of galectin-3, then one would expect to see nuclear localization of galectin-3 in both wild-type cells and cells that express the deletion mutant. The results, however, demonstrated that deletion of the galectin-3 origin domain abrogates its cellular transport to the nucleus and/or to the plasma membrane, in essence immobilizing it in the cytosol.
Studies of galectin-3 from a variety of mammalian cell types suggest that this molecule may mediate cognitive cellular interactions between cells (26) , and a role for the NH2-terminal half of galectin-3 in self-association has been suggested (27) . Deletion of the NH2-terminal half of galectin-3 leads to an extremely low self-association rate, which is essential for its bi- and multivalent functions. Mutation of galectin-3 at the collagenase cleavage site Ala62-Tyr63 diminished the lectin’s ability to induce hemagglutination by 2-fold (data not shown), whereas complete NH2-terminal deletion further abrogated the ability of galectin-3 to self-associate, diminishing its hemagglutination ability. Of interest is the previous demonstration that 11 amino acids within the extreme NH2 terminus of galectin-1 are critical for dimer formation (28) .
On the basis of the data presented here, it appears that the 11 NH2-terminal amino acids are involved in galectin-3 cellular translocation to the cell surface and to the nucleus. Ser6 point mutation abrogated phosphorylation of galectin-3 by casein kinase I at this amino acid. There were no changes in galectin-3 distribution in the mutant clones compared with wild-type galectin-3-transfected cells, suggesting that phosphorylation at this serine may not be required for galectin-3 targeting. The point mutation clones did show some changes in biological functions of galectin-3, which may not be related to cellular localization of mutant galectin-3 but may have resulted from loss of Ser6 phosphorylation. Although the precise biological mechanisms by which the NH2 terminus of galectin-3 determines function remain to be established, it may regulate molecular folding, leading to functional consequences. The observed reduction in anchorage-independent growth and tumorigenicity that results from the deletion of the 11-amino acid leader sequence motif of galectin-3 indicates that this portion of the NH2 terminus is not inert and has an important impact on both cellular localization and function that is independent of Ser6 phosphorylation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grant CA46120 (to A. R.) and NIH Grant CA69480 (to R. B.). 
2 To whom requests for reprints should be addressed, at Karmanos Cancer Institute, 110 E. Warren, Detroit, MI 48201. Phone: (313) 833-0960; Fax: (313) 831-7518; E-mail: raza{at}karmanos.org
3 The abbreviations used are: mAb, monoclonal antibody; pAb, polyclonal antibody; CMF-PBS, Ca2+-Mg2+-free PBS; GFP, green fluorescent potein.
Received 6/ 4/99. Accepted 10/15/99.
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T. Fukumori, Y. Takenaka, T. Yoshii, H.-R. C. Kim, V. Hogan, H. Inohara, S. Kagawa, and A. Raz CD29 and CD7 Mediate Galectin-3-Induced Type II T-Cell Apoptosis Cancer Res., December 1, 2003; 63(23): 8302 - 8311. [Abstract] [Full Text] [PDF]
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 C. M. John, H. Leffler, B. Kahl-Knutsson, I. Svensson, and G. A. Jarvis Truncated Galectin-3 Inhibits Tumor Growth and Metastasis in Orthotopic Nude Mouse Model of Human Breast Cancer Clin. Cancer Res., June 1, 2003; 9(6): 2374 - 2383. [Abstract] [Full Text] [PDF]
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T. Yoshii, T. Fukumori, Y. Honjo, H. Inohara, H.-R. C. Kim, and A. Raz Galectin-3 Phosphorylation Is Required for Its Anti-apoptotic Function and Cell Cycle Arrest J. Biol. Chem., February 22, 2002; 277(9): 6852 - 6857. [Abstract] [Full Text] [PDF]
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 E. Saggiorato, S. Cappia, P. De Giuli, A. Mussa, G. Pancani, P. Caraci, A. Angeli, and F. Orlandi Galectin-3 as a Presurgical Immunocytodiagnostic Marker of Minimally Invasive Follicular Thyroid Carcinoma J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5152 - 5158. [Abstract] [Full Text] [PDF]
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