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Raija Tammi - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2 and HAS3
The Journal of biological chemistry, 2015Co-Authors: Genevieve Bart, Raija Tammi, Paraskevi Heldin, Nuria Ortega Vico, Antti Hassinen, François M. Pujol, Ashik Jawahar Deen, Aino Ruusala, Anthony Squire, Sakari KellokumpuAbstract:In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.
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Extensive CD44-dependent hyaluronan coats on human bone marrow-derived mesenchymal stem cells produced by hyaluronan synthases HAS1, HAS2 and HAS3
The international journal of biochemistry & cell biology, 2014Co-Authors: Kirsi Rilla, Raija Tammi, Markku Tammi, Heikki Kröger, Mikko J. LammiAbstract:Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.
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hyaluronan synthases HAS1 3 in stromal and malignant cells correlate with breast cancer grade and predict patient survival
Breast Cancer Research and Treatment, 2014Co-Authors: Paivi Auvinen, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Ritva Tumelius, Ylermi Soini, Arto Mannermaa, Jukka Viikari, Raija TammiAbstract:Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1–3) contributes to HA accumulation. In this study, localization and expression of HAS1–3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.
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Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.
Experimental cell research, 2013Co-Authors: Hanna Siiskonen, Raija Tammi, Markku Tammi, Juha M T Hyttinen, Riikka Kärnä, Kirsi RillaAbstract:Abstract Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER–Golgi–plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.
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Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.
The Journal of biological chemistry, 2013Co-Authors: Kirsi Rilla, Raija Tammi, Juha M T Hyttinen, Tiina A Jokela, Sanna Oikari, Riikka Kärnä, Markku TammiAbstract:Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (HAS1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human HAS1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of HAS1, suggesting cellular coordination between HAS1 expression and the content of UDP-sugars.
Kirsi Rilla - One of the best experts on this subject based on the ideXlab platform.
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Extensive CD44-dependent hyaluronan coats on human bone marrow-derived mesenchymal stem cells produced by hyaluronan synthases HAS1, HAS2 and HAS3
The international journal of biochemistry & cell biology, 2014Co-Authors: Kirsi Rilla, Raija Tammi, Markku Tammi, Heikki Kröger, Mikko J. LammiAbstract:Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.
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hyaluronan synthases HAS1 3 in stromal and malignant cells correlate with breast cancer grade and predict patient survival
Breast Cancer Research and Treatment, 2014Co-Authors: Paivi Auvinen, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Ritva Tumelius, Ylermi Soini, Arto Mannermaa, Jukka Viikari, Raija TammiAbstract:Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1–3) contributes to HA accumulation. In this study, localization and expression of HAS1–3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.
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Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.
Experimental cell research, 2013Co-Authors: Hanna Siiskonen, Raija Tammi, Markku Tammi, Juha M T Hyttinen, Riikka Kärnä, Kirsi RillaAbstract:Abstract Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER–Golgi–plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.
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Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.
The Journal of biological chemistry, 2013Co-Authors: Kirsi Rilla, Raija Tammi, Juha M T Hyttinen, Tiina A Jokela, Sanna Oikari, Riikka Kärnä, Markku TammiAbstract:Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (HAS1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human HAS1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of HAS1, suggesting cellular coordination between HAS1 expression and the content of UDP-sugars.
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Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma.
BMC cancer, 2010Co-Authors: Timo K. Nykopp, Raija Tammi, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Kirsi Hämäläinen, Seppo Heinonen, Maarit AnttilaAbstract:Background Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.
Markku Tammi - One of the best experts on this subject based on the ideXlab platform.
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Extensive CD44-dependent hyaluronan coats on human bone marrow-derived mesenchymal stem cells produced by hyaluronan synthases HAS1, HAS2 and HAS3
The international journal of biochemistry & cell biology, 2014Co-Authors: Kirsi Rilla, Raija Tammi, Markku Tammi, Heikki Kröger, Mikko J. LammiAbstract:Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.
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hyaluronan synthases HAS1 3 in stromal and malignant cells correlate with breast cancer grade and predict patient survival
Breast Cancer Research and Treatment, 2014Co-Authors: Paivi Auvinen, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Ritva Tumelius, Ylermi Soini, Arto Mannermaa, Jukka Viikari, Raija TammiAbstract:Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1–3) contributes to HA accumulation. In this study, localization and expression of HAS1–3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.
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Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.
Experimental cell research, 2013Co-Authors: Hanna Siiskonen, Raija Tammi, Markku Tammi, Juha M T Hyttinen, Riikka Kärnä, Kirsi RillaAbstract:Abstract Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER–Golgi–plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.
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Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.
The Journal of biological chemistry, 2013Co-Authors: Kirsi Rilla, Raija Tammi, Juha M T Hyttinen, Tiina A Jokela, Sanna Oikari, Riikka Kärnä, Markku TammiAbstract:Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (HAS1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human HAS1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of HAS1, suggesting cellular coordination between HAS1 expression and the content of UDP-sugars.
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Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma.
BMC cancer, 2010Co-Authors: Timo K. Nykopp, Raija Tammi, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Kirsi Hämäläinen, Seppo Heinonen, Maarit AnttilaAbstract:Background Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.
Linda M Pilarski - One of the best experts on this subject based on the ideXlab platform.
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alteration of introns in a hyaluronan synthase 1 HAS1 minigene convert pre mrna splicing to the aberrant pattern in multiple myeloma mm mm patients harbor similar changes
PLOS ONE, 2013Co-Authors: Jitra Kriangkum, Andrew R Belch, Amanda Warkinton, Linda M PilarskiAbstract:Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3′splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3′ splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.
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functional and familial risk of b lineage malignancies in patients carrying single nucleotide polymorphisms snps in the hyaluronan synthase 1 gene HAS1
Blood, 2011Co-Authors: Linda M Pilarski, Jitra Kriangkum, Hemalatha Kuppusamy, Amanda Warkentin, Hlif Steingrimsdottir, Vilhelmina Haraldsdottir, James B Johnston, Sunita Ghosh, Spencer B Gibson, Helga M OgmundsdottirAbstract:Abstract 3930 Risk associations with genetic changes, especially SNPs, can be most meaningfully interpreted when the functional relevance of the involved gene(s) is known. Further, using targeted sequencing to detect SNPs provides for unequivocal allele “calling” in each patient, as well as identification of any linked low penetrance mutations that might influence risk. HAS1 is overexpressed and aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM), but not in healthy donors (HD); HAS1Vb correlated with reduced survival in a cohort of MM patients 1 . In transfectants, HAS1 variants are oncogenic in vivo and/or in vitro 2 . As shown here, a set of three intron 3 SNPs in linkage disequilbrium have significantly different genotype and allele frequencies and robust hazard ratios in people with B lineage malignancies as compared to age matched controls and to a set of unlinked exon 3 HAS1 SNPs. In all patient and control groups, all five SNPs met Hardy-Weinberg equilibrium. We sequenced an 850bp segment of the HAS1 gene (exon 3, intron 3) from PBMC of 307 Caucasian individuals, including 86 MM, 70 monoclonal gammopathy of undetermined significance (MGUS), 25 WM, 40 B chronic lymphocytic leukemia (CLL), 15 affected and 21 unaffected members of a monoclonal gammopathy prone Icelandic kindred, and 60 age-matched HD. Using direct or subclone sequencing, we evaluated the frequencies of NCBI designated minor alleles for the 5 SNPs. Bioinformatics and in vitro mutagenesis experiments confirmed that intron 3 plays a central role in clinically relevant splice site selection and aberrant intronic HAS1 splicing 3 . The linked intron 3 HAS1 SNPs (rs11084110, rs11084109 and rs11669079) in patient groups have significantly different host genotype and allele frequencies from those of HD. The frequencies of the unlinked exon 3 HAS1 polymorphisms (rs61736495, rs11084111) on the same 850bp amplicon were not significantly different between patients and HD, providing internal controls for sequencing artefacts. Associations between risk of B cell malignancy and HAS1 SNPs were evaluated using the logistic regression model. Compared to age matched controls, the HAS1 intron 3 SNPs were significantly associated with MM and MGUS for genotype frequencies (p.01 to.05) and allele frequencies (p.01 to.0007); the association was even stronger for CLL and WM (genotype frequencies p trend ranged from 4.7 to 6.8 (p=0.0001). These robust hazard ratios together with the familial analysis exclude the influence of cryptic population stratification. Risk predictions may be directly correlated with the identified SNPs, or alternatively may result from unknown mutation(s) in linkage disequilibrium with the detected SNPs. To resolve these possibilities, highly sensitive targeted sequencing provides a degree of certainty not possible with genome wide association studies. The risk associations observed here are directly correlated with the indicated SNPs because no other significant variations were identified by sequencing. The functional relevance of the associations reported here is supported by in vitro mutagenesis of intron 3, which shows a profound impact of SNPs/mutations on alternative splicing and splice site selection that leads to aberrant intronic splicing of the type seen in patients. Although multiple genes are certain to be involved, this work supports the idea that the minor alleles for the HAS1 intron 3 SNPs have a strong functional impact on epigenetic events, particularly aberrant pre-mRNA splicing, that contribute to the malignant phenotype in B lineage cancers. 1. Blood 2005 105:4836 2. JBC 2009 285:18840 3. Blood 2008 512:5111 Disclosures: Belch: Celgene: Research Funding; Onyx: Research Funding.
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clinically significant aberrant HAS1vb splicing of the hyaluronan synthase 1 gene HAS1 requires a combination of mutations and deletions in introns 3 and 4 of HAS1 minigene
Blood, 2011Co-Authors: Jitra Kriangkum, Andrew Belch, Linda M PilarskiAbstract:Abstract 3931 Clinically important aberrant splicing of the hyaluronan synthase 1 gene ( HAS1 ) occurs in malignant B cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM) patients but is undetectable in B cells from healthy donors (HD) 1 . HAS1 splice variants are generated by aberrant alternative splicing (AS) within exons 3, 4 and 5, involving exon 4 skipping (Va), partial intron 4 retention (Vc, Vd) or a combination of both (Vb). Aberrant splicing of HAS1Vb has previously shown to correlate with reduced survival in a cohort of MM patients 1 . In patients, frequent mutations have been identified in introns 3 and 4, suggesting their roles in AS 1 . To identify genetic regions involved in splice site selection leading to HAS1Vb expression, we performed in vitro sequence manipulation of introns 3 and 4. We established a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 with the upstream cDNA sequence. In an unaltered sequence of the HAS1 minigene, splicing of HAS1 full length predominated, with trace amount of variants, including the novel HAS1Vd, identified for the first time in transfectants. HAS1Vd shared an alternative acceptor site with HAS1Vb (retained 59 bp of downstream intron 4) but unlike HAS1Vb, retained exon 4. Analysis of peripheral blood mononuclear cells (PBMC) revealed that 9% of healthy donors (n=102) and MM patients (n=93) expressed HAS1Vd. About 2% coexpressed HAS1Vb and Vd. In this cohort of patients, HAS1Vb was found in 20% of unfractionated MM PBMC compared to 5% in HD PBMC, supporting our previous finding that HAS1Vb was restricted to sorted MM B cells 1 and suggesting that in HD PBMC, non-B cells are responsible for this splicing. While MM PBMCs expressed HAS1Vb>Vd, HD PBMCs and transfectants expressed HAS1Vd>Vb, indicating that splicing directed by the minigene construct was substantively different from that occurring in MM patients. In transfectants, partial deletion of HAS1 intron 4 increased HAS1Vd expression but did not affect HAS1Vb despite the fact that both variants use the same 39 splice site in intron 4. Proper joining of exons 3, 4 and 5 required a minimum of 172 bp of downstream intron 4 and acceptor site selection may be regulated by sequence between 172 and 84 bp upstream of exon 5. Thus, changes in intron 4 alone were insufficient to promote the splicing pattern observed in patients (i.e., elevated HAS1Vb). We then employed site directed mutagenesis to alter multiple G-rich regions in downstream intron 3, a strategy which enhanced exon 4 skipping as shown by increased HAS1Va expression, but did not lead to HAS1Vb splicing. Minimal manipulation to promote exon 4 skipping included base changes within the 2 tandem G-repeats (8 bp) located 73 bp upstream of exon 4, where splicing enhancer/silencer sequences were also identified. Thus, changes in intron 3 also affected the HAS1 splicing profile but like deletions in intron 4, were insufficient on their own to promote HAS1Vb expression. We next developed expression constructs that combined deletion in intron 4 with mutations in intron 3. We are able to demonstrate that when both introns were co-modified, the splicing pattern shifted towards increased HAS1Vb expression, similar to that observed in malignant cells from MM patients. Our previous work showed that deletions or mutations in the 39 end of intron 4 are frequent in MM; in silico analysis predicts splicing to form HAS1Vb 2 . To determine whether intron 3 genetic changes occur in patients, we sequenced intron 3 from genomic DNA of 50 MM PBMC. In 22/50 patients, 18 recurrent mutations unique to MM were identified in intron 3; many were also recurrent in our previous study 2 . Individual mutations recurred in 2–7 patients. Among these, 17/18 recurrent mutations increased the G-C content of intron 3 and 6/18 created or disrupted G runs in intron 3. This supports the idea that in MM patients, cumulative variations in introns 3 and 4 alter splice site selection, operationally resulting in loss of HAS1Vd and enhanced expression of the clinically relevant HAS1Vb variant. We speculate that individuals who accumulate genetic variations in introns 3 and 4 of HAS1 are predisposed to aberrant splicing of HAS1 which may contribute to development of malignancy in MM and WM. Disclosures: Belch: Celgene: Research Funding; Onyx: Research Funding.
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splicing profile of human hyaluronan synthase 1 HAS1 is altered by mutagenesis of a u ggg motifs
Blood, 2009Co-Authors: Jitra Kriangkum, Andrew R Belch, Anirban Ghosh, Amanda A Warkentin, Linda M PilarskiAbstract:Abstract 2842 Poster Board II-818 In addition to full length (FL) transcripts, clinically significant HAS 1 splice variants (Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldenstrom9s macroglobulinemia. Increased HAS1Vb expression correlates with poor survival in a cohort of MM patients. Here, we show that directed mutation of HAS1 intron 3 alters HAS1 splicing and generates a pattern of HAS1 variant expression that mimics patterns detected in MM patients. This suggests that hypermutation of HAS1 and consequent expression of HAS1 splice variants may contribute to oncogenesis in MM. HAS1FL comprises of 5 exons (2089 bp); Va skips exon 4 (133 bp); Vb skips exon 4 and partially retains 59 bp of intron upstream of exon 5 (+59); Vc has all 5 exons and partially retains 26 bp of intron downstream of exon 4. In MM, frequent intronic mutations have been observed in introns 3 and 4, suggesting possible contributions to HAS1 alternative splicing. We have utilized a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 (g345) with the upstream cDNA sequences. HAS1 expression is determined by transfection and RT-PCR using appropriated primer sets. This study focuses on identification of intronic mutations that may affect HAS1 splicing. We target mutations on (A/U)GGG motif because of its high abundance in HAS1 intron 3. The (A/U)GGG repeat was also shown to enhance the splicing of alternative intron in chicken β-tropomyosin ( Sirand-Pugnet, P, et al, NAR, 1995, 23, 3501) and intronic G runs could work in a combinatorial way to control the selection of the proper 39 splice site in human thrombopoietin ( Marcucci, R, et al, NAR, 2006, 35, 132). A 580 bp long human HAS1 intron 3 is GC-rich and comprises of 28 (A/U)GGG motifs (sequentially identified as G1, G2.., G28). HeLa cells transfected with an unmutated intron 3 construct mainly produce FL with a small amount of HAS1Va, a profile that is similar to CD40L/IL-4 activated normal B cells. Site directed mutagenesis of all 28 (A/U)GGG motifs (G1-28) abolished FL expression, but not HAS1Va, suggesting that these sequence alterations are highly unfavorable for constitutive splicing. It may be due to the loss of essential cis -acting element(s) and/or undesirable conformational changes that prevent spliceosome formation. Mutagenesis of G1-18 is shown to eliminate constitutive expression by increasing the usage of multiple alternative donor sites. Mutagenesis of G19-28 produces more HAS1Va than FL, presumably due to increased exon 4 skipping events. An increased Va/FL ratio could also be achieved by mutagenesis of G25-28 or G27-28, suggesting that this subregion is important for pathway selection. Mutagenesis was also studied in del1 construct, a unique derivative of HAS1 minigene that partially deletes intron 4. Similar to g345, del1 produces FL and HAS1Va as well as promotes expression of novel HAS1Vd, an isoform that includes +59 bp (like Vb) and exon 4. Alteration of HAS1 splicing profile caused by mutagenesis shown in g345 series is also observed in del1 series. Additionally, there is a shift from Vd to Vb expression in all constructs analysed (del1/G1-28, del1/G1-18, del1/G19-28, del1/G25-28 and del1/G27-28), a pattern of aberrant splicing that found in MM patients. Thus, in del1, increased exon 4 skipping events promote both Va and Vb expression. Sequencing of HAS1 intron 3 in a cohort of 50 MM patients indicates that recurrent mutations are found in the G repeat regions and that new repeats are generated by recurrent MM-specific HAS1 mutations. This suggests that mutation of the HAS1 construct mimics HAS1 mutation events that occur in MM patients themselves, and contributes to the clinically significant aberrant HAS1 splicing we have reported in MM ( Adamia et al. Blood, 2008, 112, 5111; Blood, 2005, 105, 4836 ). Overall, critical mutations that could alter HAS1 expression and the ratio of HAS1 variants to FL were identified in intron 3. In intron 4, critical mutations that increase the usage of alternative splice site (+59) remain to be studied. We speculate that cumulative mutations within these two intronic sequences could bring the two events together to promote HAS1Vb splicing. While trans -acting elements are likely to regulate RNA splicing and its pathway, our studies clearly suggest that intronic mutations play an important role in the aberrant splicing of human HAS1, with probable contributions to disease progression in MM. Disclosures: No relevant conflicts of interest to declare.
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aberrant splice variants of HAS1 hyaluronan synthase 1 multimerize with and modulate normally spliced HAS1 protein a potential mechanism promoting human cancer
Journal of Biological Chemistry, 2009Co-Authors: Anirban Ghosh, Hemalatha Kuppusamy, Linda M PilarskiAbstract:Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.
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Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma.
BMC cancer, 2010Co-Authors: Timo K. Nykopp, Raija Tammi, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Kirsi Hämäläinen, Seppo Heinonen, Maarit AnttilaAbstract:Background Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.
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hyaluronan synthases HAS1 3 and hyaluronidases hyal1 2 in the accumulation of hyaluronan in endometrioid endometrial carcinoma
BMC Cancer, 2010Co-Authors: Timo K. Nykopp, Raija Tammi, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Kirsi Hämäläinen, Seppo Heinonen, Maarit AnttilaAbstract:Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity. A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry. The mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001). The results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.
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expression of hyaluronan synthases HAS1 3 and hyaluronidases hyal1 2 in serous ovarian carcinomas inverse correlation between hyal1 and hyaluronan content
BMC Cancer, 2009Co-Authors: Timo K. Nykopp, Raija Tammi, Markku Tammi, Kirsi Rilla, Reijo Sironen, Veli-matti Kosma, Kirsi Hämäläinen, Annamari Heikkinen, Marja Komulainen, Maarit AnttilaAbstract:Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity. Normal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1–3 immunoreactivity in tissue sections of the same specimens. The levels of HAS2 and HAS3 mRNA (HAS1 was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of HYAL1 mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of HYAL1, but not HYAL2, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between HYAL1 mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025). The results indicate that in serous epithelial ovarian malignancies HAS expression is not consistently elevated but HYAL1 expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)
