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Tumor Biology |
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195 [N. I., T. S., K. K.], and Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Aichi 474-8522 [O. M.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HA has either directly or indirectly been implicated in a variety of cell behaviors such as adhesion, cell motility, growth, and differentiation (13, 14, 15) . Furthermore, HA-binding proteins regulate these cellular behaviors through interactions with HA and formation of HA pericellular matrix (16) . Increased matrix deposition of HA may favor tumor growth and invasion by increasing tissue hydration and by providing a suitable environment for cell migration analogous to embryonic cell movement. In addition, the HA matrix may favor tumor growth by additional mechanisms. For example, it is possible that the HA pericellular coat reduces the access of host immunocompetent cells to tumor cells (17) . In fact, a variety of tumor cells are surrounded by a thick pericellular coat that is sensitive to hyaluronidase. Removal of this coat may allow the lymphocytes to exert their cytolytic effect on the tumor cells. It has also been shown that partially degraded HA fragments promote angiogenesis, an important host contribution to tumor cell viability (18 , 19) .
Numerous animal cell mutants have been used to study the roles of glycoproteins and glycosaminoglycans in tumorigenesis and metastasis (20, 21, 22) . Therefore, HA-deficient mutant cells would provide an ideal tool for studying the relationship between HA production and the metastatic ability of cancer cells. In this study, we isolated mutants defective in the formation of HA matrix from mouse mammary carcinoma FM3A HA1 cells by treatment with a chemical mutagen, followed by selection using particle exclusion assay. All of these mutants showed marked reduction in HA production. In the present study, we compared the abilities of these mutants to form metastatic foci in lung to examine whether HA has any role in determining metastatic capacity.
Molecular cloning of the genes encoding HA synthase, which is the key enzyme in HA biosynthesis, is one of essential steps to investigate the biological functions of HA. Recently, we and other groups have isolated three mammalian HA synthase genes, HAS1, HAS2, and HAS3 (23, 24, 25) . In this study, we also demonstrated that introduction and expression of the HA synthase gene into mutant cells defective in HA biosynthesis enhanced their metastatic ability.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Particle Exclusion Assay.
Fixed sheep erythrocytes (Inter-Cell Technologies, Inc., Hopewell, NJ) were reconstituted in PBS to a density of 5 x 108 cells/ml and used for the particle exclusion assay as described previously (26)
. HA matrices were visualized by adding 1 x 107 erythrocytes to the growth medium and by viewing under an OLYMPUS IMT-2 inverted phase-contrast microscope.
Determination of HA Production by Competitive ELISA-like Assay.
The amounts of HA in culture medium were measured by a modification of the procedure described by Tengblad (27)
. We used the biotinylated HA binding region of bovine nasal cartilage proteoglycan and alkaline phosphatase-conjugated streptavidin (Amersham Pharmacia Biotech, Ltd., Uppsala, Sweden) as primary and secondary probes, respectively. The enzyme activity was measured using p-nitrophenyl phosphate (Nakalai Tesque, Inc.) as the substrate. The amounts of HA in cell layers were lower than the limit of detection, compared with that in culture medium.
HA Synthase Assays.
HA synthase activity was monitored using UDP-[14C]GlcA (272.5 mCi/mmol; NEN Life Science Products, Inc., Boston, MA) as described previously (23)
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Construction and Transfection of HAS1 Expression Vectors.
pcDNA3-HAS1 plasmid was prepared as described previously (23)
. HAS- cells were transfected either with pcDNA3-HAS1 or with pcDNA3 control vector by the lipofection procedure as described previously (23)
and then selected in the medium containing 500 µg/ml G418 (Life Technologies, Inc., Grand Island, NY). Cloned cell lines were obtained by limiting dilution.
The pIRESneo-HAS1 plasmid was made from the pIRES1neo bicistronic expression vector (Clontech Laboratories, Inc., Palo Alto, CA) and the pFLAG-HAS1 plasmid containing the mouse HAS1 cDNA. To construct a pFLAG-HAS1 plasmid, a mouse HAS1 PCR fragment was amplified using Pfu DNA polymerase (Stratagene, La Jolla, CA) and the following primers: forward, 5'-GATAGATCTGAGACAGGACATGCCAAAGCCCTCA-3' (this primer contains a BglII site and corresponds to amino acids 2RQDMPKPS of HAS1); and reverse, 5'-CACGCACCTGCGTGTTCTCACCAG-3' (corresponds to amino acids 204LVRTRRCV of HAS1). The PCR reaction conditions were as follows: 1 cycle at 94°C for 45 s, 20 cycles at 94°C for 45 s, 60°C for 45 s, 72°C for 4 min, and 1 cycle at 72°C for 10 min. The resulting PCR fragment, which includes a BspHI site, was excised at the BglII and BspHI sites and gel purified. A 3'-fragment, excised from a full-length HAS1 cDNA at BspHI and BglII sites, was also gel purified. These two HAS1 fragments were subcloned into the BglII site of pFLAG-CMV2 vector (Eastman Kodak Co., Rochester, NY) to create a pFLAG-HAS1 construct. The pFLAG-HAS1 plasmid was digested with SacI and EcoRV, and the cohesive end was repaired to blunt ends by incubation with T4 DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN). The gel-purified HAS1 cDNA fragment was subcloned into the EcoRV site of pIRES1neo vector to create the pIRESneo-HAS1 plasmid. HAS- cells were transfected either with pIRESneo-HAS1 or with pIRES1neo control vector by the lipofection procedure and then selected in the medium containing 1 mg/ml G418. The cells surviving during selection were pooled and used for the experiments.
Northern Blot Analysis and Real Time Quantitative RT-PCR Analysis.
Total RNAs were prepared using TRIZOL reagent (Life Technologies, Inc.) from exponentially proliferating FM3A HA1, mutants and transfectants. Poly(A)+ RNAs were prepared using oligo-dT cellulose (Amersham Pharmacia Biotech, Ltd.). To monitor gene expression, we used Northern blot analysis and real time quantitative RT-PCR analysis (28)
. For Northern blot, 2 µg of poly(A)+ RNA were separated by formaldehyde agarose gel electrophoresis and transferred to Hybond N+ nylon membranes (Amersham Pharmacia Biotech, Ltd.) overnight. RNAs were fixed, prehybridized, and hybridized according to the manufacturer’s recommendations. High stringency hybridization was performed using partial cDNA probes representative of mouse HAS1, mouse HAS2, mouse HAS3, and the housekeeping gene GAPDH. These probes were labeled with [
-32P]dCTP by random priming. Blots were washed to high stringency and exposed at -80°C to autoradiographic film with intensifying screens.
Real time RT-PCR analysis was performed according to the reported method (28) . Briefly, within a gene-specific PCR oligonucleotide primer pair, a fluorogenic oligonucleotide probe is designed. When the probe is intact, the reporter dye emission is quenched. During the extension phase of the PCR cycle, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe. Fluorescence intensity produced during PCR amplifications is monitored by the sequence detector directly in the reaction tube ("real time"). A computer algorithm compares the amount of reporter dye emission with the quenching dye emission and calculates the threshold cycle number (CT), when signals reach 10 times the SD of the baseline, from which amounts of various mRNAs levels of various genes tested are obtained (28) . RT-PCR reactions were performed by using TaqMan EZ RT-PCR kit (Perkin-Elmer Corp., Norwalk, CT) according to the manufacturer’s recommendations. Two hundred ng of each total RNAs were added to a 50-µl RT-PCR reaction. The reaction master mix was prepared to give final concentrations of 1x TaqMan EZ buffer, 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.6 mM dUTP, 6 mM manganese acetate, 0.01 unit/µl uracil N-glycosylase, 0.1 unit/µl rTth DNA polymerase, 200 nM concentration of the primers, and 100 nM TaqMan probe. The primers and TaqMan probe labeled with a reporter fluorescent dye (FAM) at the 5'-end and a quencher fluorescent dye (TAMRA) at the 3'-end were designed as follows: HAS1 forward primer, 5'-GGTCAGCTTCTTGAGCAGTCTT-3' (corresponding to nucleotides 891–912 of mouse HAS1 cDNA); HAS1 reverse primer, 5'-CTGTTGGCTCAACCAACGAA-3' (corresponding to the antisense complement of nucleotides 1172–1191 of HAS1); and HAS1 probe, 5'-CAGAGCTACTTCCACTGTGTGTCCTGCATC-3' (corresponding to nucleotides 949–978 of HAS1).
RT-PCR reactions and the resulting relative increase in reporter fluorescent dye emission were monitored in real time by the ABI PRISM 7700 sequence detector (Perkin-Elmer Corp., Norwalk, CT). Signals were analyzed by the sequence detector 1.0 program (Perkin-Elmer Corp.). Conditions were as follows: 1 cycle at 50°C for 2 min, 1 cycle at 60°C for 30 min, 1 cycle at 95°C for 5 min, 40 cycles at 95°C for 20 s, and 60°C for 1 min. Variation in application of sample was assessed by quantitation of GAPDH mRNA using TaqMan rodent GAPDH control reagents (Perkin-Elmer Corp.) and TaqMan EZ RT-PCR kit. The HAS1 PCR products (301 bp) amplified at the end of the reaction were analyzed by electrophoresis on 2.0% agarose gel.
Experimental Lung Metastasis.
The metastatic potentials of FM3A HA1 cells, mutants, and transfectants were determined using an "experimental metastasis assay." Cells were grown under suspension culture on 100-mm Falcon Petri dishes (no. 1005). The cells were harvested, washed three times with PBS, and suspended at 5 x 106 cells in 1 ml of HBSS. The conditioning allowed the cells to retain HA pericellular coats on their cell surfaces, as examined by flow cytometry (23)
. Male C3H/He mice, 6 weeks of age, were received i.v. injections of 0.2 ml of the cell suspension. Twenty-five days after injection, all mice were sacrificed, and their lungs were removed and fixed in Bouin’s solution. The number of lung tumor colonies was determined by counting surface colonies under a dissecting microscope.
Tumor Growth.
Male C3H/He mice, 6 weeks of age, received s.c. injections in the left flank of 0.1 ml of the cell suspension (1 x 105 cells). The tumor sizes were measured at 5-day intervals using calipers. The average of the short and long diameters was used in the determinations.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. FM3A HA1 cells were mutagenized with MNNG under strict conditions and then screened for deficiency of HA coat formation using particle exclusion assay. Five different mutant clones were isolated by limiting dilution. The clones were again confirmed to be unable to assemble the HA-rich pericellular coat. No pericellular coats were detected around the mutant cells (Fig. 1, B–F)
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. These phenotypes of the mutant cells were stable in vitro in each descendant during culture for 2 months.
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, clones HA-m2, HA-m14 (HAS-), and HA-m20 had <5% of the parental cell activity. These results suggested that the loss of HA synthase activities of these mutant cells caused the observed defects in HA production. In contrast, clones HA-m11 and HA-m12 retained >40% of the enzyme activity of the parental cells, suggesting that some aberration in the process of HA secretion or biosynthesis may be responsible for the defects observed in HA production and matrix deposition. A previous complementation experiment also demonstrated that some altered phenotypes in HA-m11 and HA-m12 cells were caused by some aberration other than that in HA synthase (23)
. We examined the growth characteristics of the parental and mutant cells in in vitro. No correlation was found between in vitro growth properties and HA production (data not shown).
Tumor Growth and Experimental Lung Metastasis of Parental and Mutant Cells.
To examine how HA production by cancer cells modifies their in vivo behavior, 1 x 105 cancer cells were injected s.c. into syngeneic mice. The animals were monitored every 5 days for tumorigenicity of the parental and mutant cells. The tumor mass in animals injected with the parental and mutant cells was first visible within 10 days. By 15 days, 100% of animals injected with parental and mutant cells had grossly visible tumors. No correlation was found between tumor growth properties and HA production (data not shown).
To evaluate the relationship between HA production and metastatic ability of cancer cells, we injected the parental and mutant cells into the tail vein of syngeneic mice and examined their lungs for tumor colonies after 25 days. The statistical significance of the apparent differences between the parental and mutant cells was tested by Student’s t test. The extent of experimental metastasis of all mutants was significantly decreased and correlated with HA production (Fig. 3)
. On the other hand, no significant differences were observed in pulmonary metastasis between the parental and MNNG-treated cells (Fig. 3)
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. In contrast, the level of expression of the HAS1 gene in HAS- cells was 0.24% of that of parental cells, as measured by real time quantitative RT-PCR analysis (Fig. 4C)
. To confirm the contribution of HA produced by cancer cells to the metastatic process, the HAS- mutant was stably transfected with the cDNA encoding mouse HAS1, which was constructed into two different types of vectors (Fig. 4A)
. Neomycin-resistant colonies arising from single cells transfected with pcDNA3-HAS1 plasmid were grown to subconfluency in 35-mm dishes and screened for HA synthetic activity. Two independent clones, HAS1-N1 and HAS1-N6, producing different levels of HA, were selected and characterized. Furthermore, neomycin-resistant cells transfected with pIRESneo-HAS1 plasmid were pooled and designated as HAS1-BS. The HAS1-N6 and HAS1-BS cells had elevated levels of HAS1 expression and HA production (Figs. 4C
and 5A
). On the other hand, HAS1-N1 had a moderate level of HAS1 expression and showed reduced HA production (Figs. 4C
and 5A
). These HAS1 transfectants also showed significant elevation in their synthase activity (Fig. 5B)
. In addition, expression of HAS1 cDNA in HAS- cells complemented the deficient matrix deposition of HA (Fig. 6)
. Mock-transfected cells were selected as controls and designated as mock (DNA3) and mock (IRES1), respectively. These mock transfectants showed comparable levels of HAS1 expression, HA production, and HA synthase activity to HAS- cells (Figs. 4C
and 5
).
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. HAS1-N6 and HAS1-BS transfectants gave rise to metastases with median numbers of 27 and 36, respectively, whereas a median number of 17 metastases was observed in the clone HAS1-N1, which showed moderate levels of HA production. The number of metastatic colonies with mock transfectants was similar to that with the HAS- cells. Taken together, these results indicate that the high HA production of mouse mammary carcinoma cells is related to their enhanced metastatic abilities.
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. These findings suggested that the difference in metastatic ability between the HAS1 and control transfectants is not primarily due to different rates of in vivo proliferation. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several mechanisms have been proposed for the involvement of HA in tumor malignancy. By analogy to embryonic cell movement, stimulated HA synthesis may favor cell growth and invasion by increasing tissue hydration and by providing a suitable environment for cell migration (1 , 29) . Toole et al. (9) observed that the HA content of invasive V2 carcinomas grown in rabbits was 3–4-fold greater than that of the same tumors grown in the nude mouse, in which it was noninvasive (9) . In addition, the HA matrix may favor tumor growth by some other mechanism. For example, the HA-rich pericellular coat constitutes a barrier that impedes both the generation of cytolytic T lymphocytes specific to antigens on the tumor cells and the lysis of tumor cells by cytolytic lymphocytes (17 , 30) . Furthermore, angiogenesis should be considered as another mechanism of HA involvement, which is vital to tumor growth and is strongly stimulated by low molecular weight HA fragments (18 , 19) .
Enhanced metastasis is the result of various cellular properties. Previous studies have shown that highly metastatic tumor cells produce higher levels of HA than low metastatic counterparts (10 , 11) . A recent pathological study also showed a positive correlation between HA content in human colorectal cancer and Dukes’ classification and suggested that the abnormal accumulation of HA in the neoplastic colon cancer provides an advantage for cancer invasion and metastasis (4) . Comparison of B16-F1 melanoma cell lines expressing different levels of cell surface HA also showed that the surface HA is correlated with metastatic behavior (12) . In addition to HA accumulation, the cell surface receptors for HA have also been implicated in the metastatic process. Overexpression of the HA receptor RHAMM caused transformation of a fibroblast cell line and induced its spontaneous metastasis in mice (31) . The observation that mutation of RHAMM in its HA-binding domains blocked ras transformation provided additional evidence connecting HA to malignant transformation. Moreover, overexpression of the specific splicing form of another HA receptor, CD44, enhanced the metastatic potential (32 , 33) . Thus, it is likely that HA is one of the key extracellular matrix molecules that control cellular events associated with cancer metastasis.
In this study, we performed genetic modification of HA by transfection of the gene encoding HA synthase. This genetic approach enabled us to control HA production and provided direct evidence for the involvement of HA in cancer metastasis. Overexpression of the HA synthase gene enhanced the metastatic potential of a mouse mammary carcinoma cell line. To our knowledge, this is the first study demonstrating that the introduction of a gene encoding HA synthase results in increased metastatic potential. However, mutant cells almost completely lacking HA production still formed a few colonies of lung metastases, suggesting that HA is not essential for metastasis. Additional studies are required to define the molecular interaction involved in HA-enhanced metastasis.
Many factors and processes are involved in cancer metastasis, i.e., penetration of surrounding tissue barriers, release of cells from the primary tumor, arrest in the microcirculation of organs, extravasation and infiltration into the stroma of these organs, and survival and growth into new tumor systems (34) . Measurements of the sizes of the s.c. transplanted tumors showed that the properties of tumor growth were similar between HAS1 and control transfectants. Zhang et al. (12) also showed that B16-F1 melanoma cells expressing different levels of cell surface HA formed s.c. tumors of approximately the same size. This result suggested that cell surface HA does not increase the growth or survival rates of cells. One possible explanation for the HA-induced enhancement of metastasis is by increased trapping of tumor cells in the capillaries of the lungs. Yoneda et al. (35) showed that lung colonization of the highly metastatic mouse mammary carcinoma cell line FM3A P15A was markedly inhibited by pretreatment of the cells with specific hyaluronidase to degrade the pericellular HA. In addition, HA oligosaccharides (>10 monosaccharide units), which are known to competitively inhibit the interaction of HA with most HA-binding molecules, significantly decreased the lung colonization of FM3A P15A cells after i.v. injection. Although the effects may be transient and reversible, it is possible that these treatments might have decreased the arrest of the microcirculation in organs and/or extravasation. Zhang et al. (12) also reported that a highly metastatic melanoma cell line expressing a high level of cell surface HA showed high-affinity binding to the SV40 virus-transformed endothelial cell line SVEC4–10 via HA-CD44 interaction. Therefore, it is likely that the interaction of the pericellular HA of cancer cells with other molecules in the circulation may be involved in high metastatic potential. In addition, we cannot rule out the possibility that HA induces cellular activation and novel gene expression through HA-receptor interaction (36 , 37) . Further investigation will be required to fully understand the mechanisms by which HA enhances the metastatic process, and genetic manipulation of HA using mammalian HA synthase genes may offer some advantage for additional studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants from the Aichi Cancer Research Foundation; by a Grant-in-Aid for Research at the Division of Matrix Glycoconjugates, Research Center for Infections Disease, Aichi Medical University, from the Ministry of Education, Culture and Science, Japan; by special coordination funds of the Science and Technology Agency of the Japanese Government; by a grant for scientific research expenses for Health and Welfare Programs; and by a special research fund from Seikagaku Corporation. 
2 To whom requests for reprints should be addressed, at Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan. Phone: 81-52-264-4811, extension 2095; Fax: 81-561-63-3532.
3 The abbreviations used are: HA, hyaluronan; MNNG, N-methyl-N'-nitro-nitrosoguanidine; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received 12/ 3/98. Accepted 3/19/99.
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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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N. Liu, F. Gao, Z. Han, X. Xu, C. B. Underhill, and L. Zhang Hyaluronan Synthase 3 Overexpression Promotes the Growth of TSU Prostate Cancer Cells Cancer Res., July 1, 2001; 61(13): 5207 - 5214. [Abstract] [Full Text] [PDF]
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 Y. Sohara, N. Ishiguro, K. Machida, H. Kurata, A. A. Thant, T. Senga, S. Matsuda, K. Kimata, H. Iwata, and M. Hamaguchi Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen-activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner Mol. Biol. Cell, June 1, 2001; 12(6): 1859 - 1868. [Abstract] [Full Text] [PDF]
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A. Suzuki, H. Toyoda, T. Toida,, and T. Imanari Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides Glycobiology, January 1, 2001; 11(1): 57 - 64. [Abstract] [Full Text] [PDF]
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R. M. Peterson, Q. Yu, I. Stamenkovic, and B. P. Toole Perturbation of Hyaluronan Interactions by Soluble CD44 Inhibits Growth of Murine Mammary Carcinoma Cells in Ascites Am. J. Pathol., June 1, 2000; 156(6): 2159 - 2167. [Abstract] [Full Text] [PDF]
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 S. Oliferenko, I. Kaverina, J. V. Small, and L. A. Huber Hyaluronic Acid (Ha) Binding to Cd44 Activates Rac1 and Induces Lamellipodia Outgrowth J. Cell Biol., March 20, 2000; 148(6): 1159 - 1164. [Abstract] [Full Text] [PDF]
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M. A. Anttila, R. H. Tammi, M. I. Tammi, K. J. Syrjänen, S. V. Saarikoski, and V.-M. Kosma High Levels of Stromal Hyaluronan Predict Poor Disease Outcome in Epithelial Ovarian Cancer Cancer Res., January 1, 2000; 60(1): 150 - 155. [Abstract] [Full Text]
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N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, et al. Three Isoforms of Mammalian Hyaluronan Synthases Have Distinct Enzymatic Properties J. Biol. Chem., August 27, 1999; 274(35): 25085 - 25092. [Abstract] [Full Text] [PDF]
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L. Huang, N. Grammatikakis, M. Yoneda, S. D. Banerjee, and B. P. Toole Molecular Characterization of a Novel Intracellular Hyaluronan-binding Protein J. Biol. Chem., September 15, 2000; 275(38): 29829 - 29839. [Abstract] [Full Text] [PDF]
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M. A. Simpson, J. Reiland, S. R. Burger, L. T. Furcht, A. P. Spicer, T. R. Oegema Jr., and J. B. McCarthy Hyaluronan Synthase Elevation in Metastatic Prostate Carcinoma Cells Correlates with Hyaluronan Surface Retention, a Prerequisite for Rapid Adhesion to Bone Marrow Endothelial Cells J. Biol. Chem., May 18, 2001; 276(21): 17949 - 17957. [Abstract] [Full Text] [PDF]
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