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Tumor Biology |
Department of Oncology, Lombardi Cancer Center, Georgetown University Medical School, Washington DC 20007
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At present, three closely related isoforms for HAS3 have been described, termed HAS1, 2, and 3, each of which appears to be associated with the plasma membrane (12, 13, 14, 15, 16, 17, 18, 19) . They have a predicted molecular mass of approximately 63 kDa, and transfection of cells with expression vectors for each of these isoforms can induce the synthesis of hyaluronan and the formation of a pericellular coat. However, the three isoforms are distinguished from each other with respect to: (a) their expression pattern during embryonic development and distribution in adult tissues; (b) the phenotype of knockout mutants in mice in which loss of HAS2 resulted in an embryonic lethality, whereas ones deficient in HAS1 and HAS3 were viable and had no obvious phenotype; (c) the rate at which they carry out hyaluronan synthesis, with HAS3 being more active than either HAS1 and HAS2; and (d) the size of the hyaluronan produced by the different isoforms with HAS3 giving rise to a somewhat smaller product than either HAS1 or 2 (17 , 18) .
Several recent studies have shown that transfection of tumor cells with expression vectors for hyaluronan synthase alters their behavior; e.g., Kosaki et al. (20) reported that the transfection of human fibrosarcoma cells with HAS2 enhanced both anchorage-independent growth and tumorigenicity. In addition, Itano et al. (21) have found that clones of mouse mammary cancer cells that had low levels of hyaluronan synthesis demonstrated decreased metastatic properties, which could be restored if the cells were transfected with an expression vector for HAS1.
In this study, we have cloned and characterized the full-length cDNA for human HAS3 and examined its potential role in tumor progression. An expression vector carrying HAS3 was transfected into TSU prostate cancer cells, and alterations in the phenotype of the resulting cells were examined. We found that the HAS3-induced overexpression of hyaluronan enhanced the rate of tumor cell growth both in vitro and in vivo. Xenografts of the HAS3-transfected cells grew faster and larger, demonstrated a hyaluronan-rich stroma and increased vascularization. However, transfection with HAS3 did not shift the TSU cells to a more metastatic phenotype, suggesting that hyaluronan alone is not sufficient for metastasis in the case of TSU cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and Characterization of cDNA for Human HAS3.
Oligonucleotide primers for PCR of a partial sequence of cDNA for human HAS3 were designed according to the published sequence of Spicer and McDonald (Ref. 17
; GenBank accession no. U86409) and consisted of the following: 5'-TCCTACTTTGGCTGTGTGCAG and 3'-AGATTTGTTGATGGTAGCAAT. A human brain cDNA library (Clontech, Palo Alto, CA) was used as a template, and PCR was performed to generate a 570-bp fragment of human HAS3 cDNA. This fragment was then used to probe the human brain cDNA library. Positive clones were isolated, amplified, and sequenced. The Mac-Vector program was used to carry out homologue analysis of the cloned human HAS3 with other human and mouse HASs.
Construction and Transfection of Human HAS3 Expression Vector.
The cDNA for HAS3 was subcloned into the pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA), and correct clones were identified by restriction endonuclease map analysis. The HAS3-pcDNA3 or pcDNA3 (control vector) were transfected into TSU cells using the calcium precipitation method (22)
. The clones that survived in 1 mg/ml of Geneticin (G418) (>100 individual clones in each case) were pooled and expanded for further characterization.
Characterization of Transfected Cells.
The presence of HAS3 message was determined by RT-PCR using the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ). The primers consisted of the following: 5'-TCATGGTGGTGGATGGCAACCGC and 3'-CTAAGCCACCTGATGTACGTCCA, which gave rise to a 283-bp reaction product with HAS3 and a 324-bp product with HAS1. The amplified sequences were analyzed by agarose gel electrophoresis followed by staining with ethidium bromide.
Two methods were used to determine hyaluronan production by the transfected cells. The first consisted of dot blot analysis of secreted hyaluronan, in which conditioned media from vector-TSU and HAS3-TSU cells that had been cultured at similar densities for 3 days were applied to nitrocellulose membrane using a dot blot apparatus. After washing with PBS containing 0.1% Tween 20, the membrane was blocked with 5% nonfat milk and 1% polyvinylpyrrolidone in PBS for 30 min. The hyaluronan was detected by sequential incubations in 1 µg/ml of b-HABP (23) for 1 h, 0.25 µg/ml of peroxidase-labeled streptavidin for 1 h, and finally a chemiluminescent substrate for peroxidase.
The second method for quantitation of hyaluronan consisted of a modified enzyme-linked assay (23) . For this assay, a high-bound ELISA plate (Falcon, Lincoln Park, NJ) was coated with 100 µg/ml of crude human umbilical cord hyaluronan (Sigma Chemical Co., St. Louis, MO) in PBS at room temperature overnight and blocked with 10% calf serum, 90% PBS. The samples of conditioned media and cell lysates were adjusted to equal protein concentrations, and 25-µl samples were mixed with 100 µl of 50 µg/ml of b-HABP at 37°C for 1 h and then transferred to the hyaluronan-coated ELISA plate. The unbound b-HABP remaining in the sample mixture could then bind to the hyaluronan-coated plate and was detected by incubation with 0.5 µg/ml of peroxidase-labeled streptavidin followed by a peroxidase substrate consisting of H2O2 and azinobis (3-ethyl-benzthiazoline sulfonic acid) in 0.1 M Na citrate (pH 5.0). The plate was read at A405, and the concentration of hyaluronan in the samples was calculated from a standard curve.
The expression of CD44 by the vector-TSU and HAS3-TSU cells was examined by Western blotting. For this, both low- and high-density cultures were harvested with EDTA in PBS, and equivalent amounts of protein were dissolved in Laemmli sample buffer under nonreducing conditions and electrophoresed on a 10% SDS polyacrylamide gel. The resulting gel was electrophoretically transferred to a sheet of nitrocellulose and stained for CD44 using the BU52 monoclonal antibody (The Binding Site, Birmingham, United Kingdom) as described previously (24) .
Anchorage-dependent Growth.
Aliquots of medium (10% calf serum, 90% DMEM) containing 5000 transfected cells were added to 24-well dishes. At various times, the cells were harvested in 5 mM EDTA in PBS, and the cell number was determined with a Coulter counter. In some experiments, the cells were grown in the presence of 2 to 200 µg/ml of high molecular weight hyaluronan (Lifecore Biomedical, Chaska, MN) for 7 days.
Colony Formation.
Vector-TSU or HAS3-TSU cells (20,000) were suspended in 1 ml of 0.36% agarose, 10% fetal bovine serum, 90% DMEM and then immediately placed on top of a layer of 0.6% solid agarose with 10% fetal bovine serum, 90% DMEM. Two weeks later, the number of colonies larger than 50 µm in diameter was quantified using an Omnicon Image Analysis system (Imaging Products International, Chantilly, VA).
Endothelial Cell Proliferation and Migration.
For the proliferation assay, 2 x 103 ABAE cells were subcultured into 96-well plates and allowed to grow overnight. The next day, the media was replaced with 150 µl of 1% calf serum, 99% DMEM along with 50 µl of conditioned medium from either vector-TSU or HAS3-TSU cells. After 36 h, 0.3 µCi of [3H]thymidine was added to each well, and 8 h later the cells were processed with an autoharvester. The incorporated [3H]thymidine was determined with a ß-counter.
For the migration assay, 25-µl aliquots of 10% FCS, 90% DMEM containing 5 x 103 ABAE cells were added to bottom wells of a 48-well Boyden chamber (Nucleopore, Pleasanton, CA) and then covered with a nucleopore membrane (5-µm pore size) coated with 0.1 mg/ml gelatin (25) . The top well chamber was assembled and inverted for 2 h to allow the cells to adhere to the bottom side of membrane and then turned upright. Conditioned medium (50 µl) from either the vector-TSU or HAS3-TSU cells was added to top wells of the chamber and incubated for 2 h. The membrane was removed, the bottom side was carefully wiped to remove cells that had not migrated, and then the cells on the topside were stained with Hema 3 (Biochemical Science, Swedenboro, NJ). The membrane was placed on a slide, and the number of cells that had migrated to the topside were counted in 10 random high-powered fields.
Tumor Growth and Metastasis Assay.
Two assay systems were used to determine tumor growth in vivo. In the first assay, 2 x 106 vector-TSU and HAS3-TSU cells were placed on the chorioallantoic membranes of 10-day-old chicken embryos (15 eggs/group) and incubated at 37°C for 5 days. The tumor masses that grew on the chorioallantoic membranes were removed, photographed, and weighed. In the second assay system, the transfected cells were injected s.c. into 5-week-old male nude mice (2 x 106 cells/site; five mice/group), and the size of the xenografts was measured twice a week. At the end of 3 weeks, the mice were sacrificed, and the xenografts were photographed, weighted, and then fixed with 3.7% formaldehyde or frozen in liquid nitrogen for immunohistochemical staining.
To examine experimental metastasis, 5 x 105 vector-TSU and HAS3-TSU cells were injected into the tail veins of 5-week-old nude mice (five mice/group). Three weeks later, the mice were sacrificed, and the lungs were examined under a dissecting microscope for metastases.
Histological Staining.
To stain cultured cells for hyaluronan, the transfected cells were seeded into an 8-well chamber slide (Nunc, Naperville, IL) and allowed to grow to confluence. After fixation in 3.7% formaldehyde for 5 min, the cells were washed and stained with 10 µg/ml b-HABP in 10% calf serum, 90% PBS, followed by 4 µg/ml of peroxidase-conjugated streptavidin and finally a substrate consisting of 3-amino-9-ethyl-carbazole and H2O2 that gives a dark red reaction product (23)
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To stain xenografts for hyaluronan, the tissue was fixed with formaldehyde, embedded in paraffin, and cut into 5-µm thick sections. After the removal of the paraffin, the sections were processed as described above. After the immunoperoxidase reaction step, the sections were counter-stained with Mayer’s hematoxylin and then preserved with Crystal/Mount (Biomeda, Foster City, CA).
To stain for endothelial cells, samples of the xenografts were rapidly frozen, cut into 10-µm thick sections, fixed in acetone, and air-dried. The sections were incubated sequentially with: (a) a 1:30 dilution of rat antimouse CD31 (PharMigen, San Diego, CA) in 10% calf serum, 90% PBS for 1 h; (b) avidin-biotin complex method reagents for rat IgG (ABC kits; Biomeda); (c) a peroxidase substrate consisting of H2O2 and 3-amino-9-ethyl-carbazole; and (d) counter-stained with Mayer’s hematoxylin. The numbers of immunopositive spots corresponding to small blood vessels were counted in 10 random fields of three samples from each group.
Statistical Analysis.
The mean and SE were calculated from the raw data and then subjected to Student’s t test. The P < 0.05 was regarded as statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The HAS3 protein had a deduced molecular weight of about 63,000 and a pI of 8.70. The first 27 amino acids represented the signal peptide (the cleavage site is between ILA and AY). There were six transmembrane sequences, one in the NH2 terminus and five in the COOH terminus. Between the first transmembrane sequence (from amino acid 43 to 65) and second transmembrane sequence (from amino acid 384 to 402), there was a stretch of 319 amino acids located outside of plasma membrane, which appeared to be the major functional region for polysaccharide synthesis. The 170 amino acids in the COOH terminus (from amino acid 384 to 553) contained five transmembrane domains that can form loops spanning in and out of the plasma membrane. A potential N-glycosylation site was present at amino acid position 462, a glycosaminoglycan attachment site at position 464, and several phosphorylation sites for tyrosine kinase, casein kinase, protein kinase C, and cyclic AMP- and cyclic GMP-dependent protein kinases. HAS3 also contained seven hyaluronan-binding motifs of B(X7)B in which B is either R or K, and X7 contains no acidic residues and at least one basic amino acid (26)
. Similar domains are present in other hyaluronan-binding proteins such as RHAMM, CD44, hyaluronidase, link protein, aggrecan, human GHAP, and TSG-6. Fig. 2
shows that compared with related enzymes, human HAS3 was about 53% identical to HAS1 (both human and mouse forms), 67% identical to HAS2, and 96% identical to mouse HAS3. These results are consistent with earlier studies of this and related genes (13, 14, 15, 16, 17, 18, 19)
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, the HAS3-TSU cells gave rise to a PCR product of approximately 280 bp corresponding to HAS3, whereas no such band was detected in the vector-TSU cells. In addition, no reaction product of 314 bp was apparent, indicating that the message for HAS1 was absent from these cells.
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, conditioned medium from HAS3-TSU cells contained a significantly greater amount of hyaluronan than that from vector-TSU cells. To quantitatively measure the hyaluronan, a competitive enzyme-linked assay was performed. Fig. 3, C and D
show that both conditioned medium and lysates of HAS3-TSU cells contained greater amounts of hyaluronan as compared with those of the control vector-TSU cells. However, there was no obvious difference in the level of CD44, a cell surface receptor for hyaluronan, on the two cell types at either low or high density as determined by Western blotting (data not shown). Taken together, these results indicate that the transfection of TSU cells with cDNA for HAS3 stimulated their production of hyaluronan.
HAS3 Promotes Cell Growth at High Densities.
We then compared the growth rates of the vector-TSU and HAS3-TSU cells. For this, the transfected cells were subcultured at similar starting densities, and at various times thereafter the cells were harvested and enumerated with a Coulter counter. Fig. 4A
shows that during the first 4 days, there was no obvious difference in the proliferation rates of the vector-TSU and HAS3-TSU cells. However, after day 5, the HAS3-TSU cells began to proliferate at a faster rate than the vector-TSU cells. Thus, at high densities, the HAS3-transfected cells grew at a faster rate. This conclusion was also suggested by our recent finding that transfection of MDA-231 human breast cancer cells with antisense to HAS3 results in decreased expression of hyaluronan and inhibition of their growth at high densities.4
Together, these results indicate that HAS3 plays a role in cell proliferation at high densities.
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(top), the vector-TSU cells displayed a cobblestone appearance indicative of contact inhibition of growth with relatively little staining of pericellular hyaluronan. In contrast, the HAS3-TSU cells (Fig. 4B
, bottom) appeared to have lost contact-inhibition of growth, forming numerous multilayered clusters of cells, which were associated with most of the hyaluronan staining. In addition, the HAS3-TSU cells displayed an enhanced ability to form colonies in soft agar as compared with the vector-TSU cells (Fig. 4C)
. Thus, at high densities, the HAS3-transfected cells grew at a faster rate, presumably because they had partially lost contact inhibition of growth.
Conditioned Medium from HAS3-TSU Stimulated the Proliferation and Migration of Endothelial Cells.
Because several studies (27, 28, 29)
reported that hyaluronan can modulate angiogenesis, we examined the effects of conditioned medium from HAS3-TSU cells on the behavior of cultured endothelial cells. When conditioned medium from HAS3-TSU cells was added to the medium of ABAE cells, it stimulated their proliferation by 66% as compared with that from vector-TSU cells (Fig. 5A)
. Furthermore, conditioned medium from the HAS3-TSU also stimulated the migration of ABAE cells through nucleopore filters by more than 300% as compared with conditioned medium from control cells (Fig. 5B)
. These results further suggest that the hyaluronan produced by HAS3-TSU cells could exert a stimulatory effect on endothelial cells.
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, the HAS3-TSU xenografts were significantly larger than those of the vector-TSU cells. Histological examination of the xenografts revealed that, whereas the vector-TSU cells were compact, the HAS3-TSU cells were more dispersed with increased intercellular spaces (data not shown). These results suggested that overexpression of hyaluronan enhanced the tumor growth on the chorioallantoic membrane system.
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, suggesting that HAS3 promotes the growth of TSU tumor cells in mice. These results are consistent with those obtained from the chicken chorioallantoic membrane system.
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Increased Extracellular Hyaluronan and Angiogenesis in HAS3-TSU Xenografts.
The xenografts from nude mice were processed for histology, and the resulting sections were stained for hyaluronan (Fig. 8A)
. Although the xenografts varied from region to region, in general, the cells in the vector-TSU xenografts were relatively homogeneous and compact, and most of the hyaluronan appeared to be present in the cytoplasm of the cells (Fig. 8A)
. In contrast, the HAS3-TSU cells formed small clusters or nests of cells that were surrounded by a matrix rich in hyaluronan (Fig. 8A)
. Such structures were not observed in the control-TSU xenografts.
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shows that there was strong positive staining in HAS3-TSU xenografts as compared with the control vector-TSU xenografts. The number of vessels in 10–15 random fields was significantly higher in the HAS3-TSU xenografts than in the control xenografts (Fig. 8C)
. This suggests that increased levels of hyaluronan can stimulate angiogenesis in mice, and this may, in part, account for the faster growth rate of tumors formed by HAS3-TSU cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study also suggested that stimulation of hyaluronan synthesis in TSU cells by transfecting them with HAS3 expression vectors enhanced their growth both in chicken embryos and in nude mice. This enhanced tumor growth appeared to be attributable to two distinct mechanisms. The first involved a direct effect on the tumor cells themselves, as indicated by the fact that in tissue culture, HAS3-transfected cells grew at a faster rate at high density. The second mechanism promoting tumor growth rate resulted from an increase in vascularization, as reflected by the greater density of blood vessels in xenografts from nude mice. Together, these two factors contribute to the increased tumor growth rate.
At present, we believe that the effects of HAS3 transfection on TSU cell phenotype are a direct consequence of increased hyaluronan synthesis. Along these lines, it is important to note that HAS3 overexpression increases the production of both secreted and cell-associated hyaluronan. These two pools of hyaluronan may have different effects on cell behavior. This was suggested by preliminary experiments in which we found that the addition of high molecular weight hyaluronan to cultures of vector-TSU or HAS3-TSU cells had no obvious effect on their growth rates (data not shown). We believe that under these specific conditions, free hyaluronan of high molecular weight did not affect the behavior of these cells. Rather, we believe that it was the cell-associated fraction of hyaluronan induced by HAS3 that played a more important role in stimulating cell growth. However, we cannot eliminate the possibility that hyaluronan of the appropriate size and concentration may indeed influence the behavior of TSU cells, similar to the effects that it has on endothelial cells (27 , 28) . It is also possible that HAS3 may have effects on the TSU cells independent of its function to promote hyaluronan synthesis; e.g., HAS3 could influence the interaction of the plasma membrane with elements of the cytoskeleton and thereby alter cell behavior.
The hyaluronan on the surface of cultured cells can form a pericellular coat that can be directly visualized by its ability to exclude small particles such as erythrocytes (30) . In the case of rat fibrosarcoma cells, this coat is composed of small, microvilli-like projections that extend out from the surface of the cells to which the hyaluronan is attached (31) . This pericellular coat could stimulate the growth of cells by several different mechanisms. One possible mechanism is that it disrupts intercellular junctions and thereby allows the cells to detach from the substrate so that they can divide and occupy new space (32, 33, 34) . This would allow cells to overcome contact inhibition of growth that is characteristic of TSU cells and allow them to from multilayers at high-density cultures as we have observed. Another possibility is that the hyaluronan interacts with receptors on the surfaces of cells such as CD44 or RHAMM to influence their migratory and proliferative behavior (35 , 36) .
Extracellular hyaluronan can also stimulate cell proliferation by increasing the flow of nutrients. Indeed, the extracellular hyaluronan apparent in xenografts of HAS3-TSU cells in nude mice could serve as conduits through which nutrients diffuse to support cells located some distance from the blood supply and thus facilitate their growth. In the xenografts of vector-TSU cells that lacked these hyaluronan rich spaces, the tumor cells formed a continuous mass and were more susceptible to necrosis. Along these lines, extracellular hyaluronan is prominent in the lower regions of most, if not all, stratified epithelium, where it is believed to maintain spaces so that nutrients can diffuse to the more superficial epithelial cells (37 , 38) .
Hyaluronan also appeared to promote vascularization that is clearly important in regulating tumor growth (39 , 40) . This was indicated by our observation that xenografts of HAS3-TSU cells formed in nude mice had a greater density of blood vessels than did control xenografts. Part of this increased vascularization may be attributable to the pericellular spaces generated by the hyaluronan that provides space that facilitates the invasion of endothelial cells. In addition, the hyaluronan itself can stimulate the migration of endothelial cells. This was also indirectly suggested by experiments showing that conditioned media from the HAS3-transfected cells stimulated the growth and migration of cultured endothelial cells. This is consistent with the earlier studies of West et al. (27 , 28) who have shown that oligosaccharide fragments of hyaluronan stimulated the formation of new blood vessels in the chorioallantoic membrane of chicken embryos.
Although the results of this study suggest that overexpression of HAS3 in TSU prostate cancer cells promotes their tumorigenicity, there are some aspects that appear to contradict the findings of other studies; e.g., although we found that TSU transfectants grew faster in culture, Kosaki et al. (20) found no such increase in the growth of HT1080 cells transfected with HAS2 under anchorage-dependent conditions; however, these cells did form larger colonies in suspension culture. We believe that this difference may be attributable in part to the different target cells that were used in these studies. The TSU cells used as targets in this study are of epithelial origin, whereas HT1080 cells are derived from a fibrosarcoma of connective tissue cells. This could also account for the differences seen with regard to growth behavior and the production of hyaluronan in the connective tissue and their ability to stimulate angiogenesis. Alternatively, the differences could be attributed to the characteristics of the particular HASs that were used in these studies, because they differ with respect to both their synthetic activity as well as the size of the hyaluronan that they produce (16, 17, 18) .
Another apparent discrepancy was our observation that transfection of TSU cells with HAS3 did not appear to stimulate their ability to form lung metastases in nude mice. In contrast, we had reported previously (3) that the levels of hyaluronan on the surface of B16 cells were directly correlated with their metastatic behavior. Similarly, Itano et al. (21) found that transfection of FM3A with HAS1 enhanced their metastatic properties. Again, we believe that these divergent results were attributable to the different cell types that were used as targets for transfection. In the case of B16 and FM3A, these cell lines originally possessed the ability to undergo metastasis, and stimulation of hyaluronan synthesis in these cells enhanced this innate property. In contrast, TSU cells appeared to lack this ability (at least in nude mice), and increased hyaluronan synthesis, by itself, is not sufficient to promote metastatic properties. Clearly, the process of metastasis is a complex phenomenon involving the collaboration of many molecules. Although the production of hyaluronan is one of the factors, it is not sufficient for tumor metastasis in the case of TSU cells.
In conclusion, the results of this study indicate that HAS3 expression plays a role in tumor progression and are consistent with earlier studies demonstrating a correlation between hyaluronan levels and tumorigenicity. Furthermore, the hyaluronan may be acting through several different mechanisms, including: (a) a direct effect on the growth of the tumor cells themselves; (b) the formation of extracellular conduits through which nutrients can flow; and (c) the stimulation of blood vessel growth. However, these effects depend upon the particular cell type and the specific environment. Given the complexity of the effects of hyaluronan, it may be difficult to predict exactly how it will influence the behavior of tumor cells. In the case of TSU cells, hyaluronan may be more of a facilitator of tumor growth rather than an instigator of metastasis.
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FOOTNOTES |
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1 Supported in part by National Cancer Institute/NIH (R29 CA71545), United States Army Medical Research and Materiel Command (DAMD 17-99-1-9031; DAMD 17-98-1-8099; PC970502) and Susan G. Komen Breast Cancer Foundation (to L. Z. and C. B. U.). The animal protocols used in this study were approved by the Georgetown University Animal Care and Use Committee. 
2 To whom requests for reprints should be addressed, at Department of Oncology, Lombardi Cancer Center, Georgetown University Medical School, 3970 Reservoir Road, NW, Washington DC 20007. Phone: (202) 687-6397; Fax: (202) 687-7505; E-mail: Zhangl{at}gusun.georgetown.edu
3 The abbreviations used are: HAS, hyaluronan synthase; ABAE, adult bovine aorta endothelial cells; b-HABP, biotinylated hyaluronan-binding protein from cartilage; RT-PCR, reverse transcription-polymerase chain reaction.
Received 12/18/00. Accepted 4/25/01.
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I. Kakizaki, K. Kojima, K. Takagaki, M. Endo, R. Kannagi, M. Ito, Y. Maruo, H. Sato, T. Yasuda, S. Mita, et al. A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone J. Biol. Chem., August 6, 2004; 279(32): 33281 - 33289. [Abstract] [Full Text] [PDF]
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H.-R. Kim, M. A. Wheeler, C. M. Wilson, J. Iida, D. Eng, M. A. Simpson, J. B. McCarthy, and K. M. Bullard Hyaluronan Facilitates Invasion of Colon Carcinoma Cells in Vitro via Interaction with CD44 Cancer Res., July 1, 2004; 64(13): 4569 - 4576. [Abstract] [Full Text] [PDF]
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N. Itano, T. Sawai, F. Atsumi, O. Miyaishi, S. Taniguchi, R. Kannagi, M. Hamaguchi, and K. Kimata Selective Expression and Functional Characteristics of Three Mammalian Hyaluronan Synthases in Oncogenic Malignant Transformation J. Biol. Chem., April 30, 2004; 279(18): 18679 - 18687. [Abstract] [Full Text] [PDF]
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 K. M. Stuhlmeier and C. Pollaschek Glucocorticoids inhibit induced and non-induced mRNA accumulation of genes encoding hyaluronan synthases (HAS): hydrocortisone inhibits HAS1 activation by blocking the p38 mitogen-activated protein kinase signalling pathway Rheumatology, February 1, 2004; 43(2): 164 - 169. [Abstract] [Full Text] [PDF]
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A. Zoltan-Jones, L. Huang, S. Ghatak, and B. P. Toole Elevated Hyaluronan Production Induces Mesenchymal and Transformed Properties in Epithelial Cells J. Biol. Chem., November 14, 2003; 278(46): 45801 - 45810. [Abstract] [Full Text] [PDF]
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 S. Desrivieres, T. Prinz, N. Castro-Palomino Laria, M. Meyer, G. Boehm, U. Bauer, J. Schafer, T. Neumann, C. Shemanko, and B. Groner Comparative Proteomic Analysis of Proliferating and Functionally Differentiated Mammary Epithelial Cells Mol. Cell. Proteomics, October 1, 2003; 2(10): 1039 - 1054. [Abstract] [Full Text] [PDF]
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S. Ghatak, S. Misra, and B. P. Toole Hyaluronan Oligosaccharides Inhibit Anchorage-independent Growth of Tumor Cells by Suppressing the Phosphoinositide 3-Kinase/Akt Cell Survival Pathway J. Biol. Chem., October 4, 2002; 277(41): 38013 - 38020. [Abstract] [Full Text] [PDF]
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B. P. Toole and V. C. Hascall Hyaluronan and Tumor Growth Am. J. Pathol., September 1, 2002; 161(3): 745 - 747. [Full Text] [PDF]
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M. A. Simpson, C. M. Wilson, and J. B. McCarthy Inhibition of Prostate Tumor Cell Hyaluronan Synthesis Impairs Subcutaneous Growth and Vascularization in Immunocompromised Mice Am. J. Pathol., September 1, 2002; 161(3): 849 - 857. [Abstract] [Full Text] [PDF]
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 N. Itano, F. Atsumi, T. Sawai, Y. Yamada, O. Miyaishi, T. Senga, M. Hamaguchi, and K. Kimata Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration PNAS, March 19, 2002; 99(6): 3609 - 3614. [Abstract] [Full Text] [PDF]
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M. A. Simpson, C. M. Wilson, L. T. Furcht, A. P. Spicer, T. R. Oegema Jr., and J. B. McCarthy Manipulation of Hyaluronan Synthase Expression in Prostate Adenocarcinoma Cells Alters Pericellular Matrix Retention and Adhesion to Bone Marrow Endothelial Cells J. Biol. Chem., March 15, 2002; 277(12): 10050 - 10057. [Abstract] [Full Text] [PDF]
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 B. P. Toole Hyaluronan promotes the malignant phenotype Glycobiology, March 1, 2002; 12(3): 37R - 42R. [Abstract] [Full Text] [PDF]
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B. P. Toole, T. N. Wight, and M. I. Tammi Hyaluronan-Cell Interactions in Cancer and Vascular Disease J. Biol. Chem., February 8, 2002; 277(7): 4593 - 4596. [Full Text] [PDF]
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