HYAL4-V1/Chondroitinase (Chase) Drives Gemcitabine Resistance and Predicts Chemotherapy Failure in Patients with Bladder Cancer.

PURPOSE: Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers. Because of better tolerability, gemcitabine + cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer. Nevertheless, predicting treatment failure and overcoming resistance remain unmet clinical needs. We discovered that splice variant (V1) of HYAL-4 is a first-in-class eukaryotic chondroitinase (Chase), and CD44 is its major substrate. V1 is upregulated in bladder cancer and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance. EXPERIMENTAL DESIGN: V1 expression was measured in muscle-invasive bladder cancer (MIBC) specimens by qRT-PCR and IHC. HYAL-4 wild-type (Wt) and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for chemoresistance and associated mechanism in preclinical models. RESULTS: V1 levels in MIBC specimens of patients who developed metastasis, predicted response to gemcitabine + cisplatin adjuvant/salvage treatment and disease-specific mortality. V1-expressing bladder cells were resistant to gemcitabine but not to cisplatin. V1 expression neither affected gemcitabine influx nor the drug-efflux transporters. Instead, V1 increased gemcitabine metabolism and subsequent efflux of difluorodeoxyuridine, by upregulating cytidine deaminase (CDA) expression through increased CD44-JAK2/STAT3 signaling. CDA inhibitor tetrahydrouridine resensitized V1-expressing cells to gemcitabine. While gemcitabine (25-50 mg/kg) inhibited bladder cancer xenograft growth, V1-expressing tumors were resistant. Low-dose combination of gemcitabine and tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity. CONCLUSIONS: V1/Chase drives gemcitabine resistance and potentially predicts gemcitabine + cisplatin failure. CDA inhibition resensitizes V1-expressing tumors to gemcitabine. Because several chemotherapy regimens include gemcitabine, our study could have broad significance.

In Vivo Degradation of Crosslinked Hyaluronic Acid Fillers by Exogenous Hyaluronidases.

BACKGROUND: An advantage of hyaluronic acid (HA)-based fillers is reversibility. OBJECTIVE: To evaluate the ability of 2 hyaluronidases to degrade 3 HA-based fillers using a novel in vivo model. MATERIALS AND METHODS: Rats were injected with 3 HA fillers (HYC-24L+, VYC-20L, and RES-L) to create a projecting bolus. After 4 days, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) was administered at (1) varying doses (5 U, 10 U, or 30 U per 0.1 mL filler) or (2) different dilutions (10 U diluted 3-fold). The impact of tissue integration was assessed by administering 10 U/0.1 mL filler 4 weeks after filler injection. Three-dimensional images quantified projection loss over 72 hours. RESULTS: Complete loss of projection was achieved for all fillers with the highest HX and VIT doses; lower doses achieved less degradation. No difference in degradation was observed between HYC-24L+ and VYC-20L using HX or VIT. RES-L was slightly more degraded with 10 U VIT but not with 10 U HX. Enzyme dilution resulted in less degradation. Tissue integration did not impact the degree of degradation. CONCLUSION: This model incorporates the biological system while controlling variables including filler depth and volume and location of hyaluronidase delivery. Hyaluronic acid filler degradation by exogenous hyaluronidase was not hindered by differences among fillers.

Histochemical structure and immunolocalisation of the hyaluronan system in the dromedary oviduct.

We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.

Hyaluronidase 2: a novel germ cell hyaluronidase with epididymal expression and functional roles in mammalian sperm.

To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ~57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization.

Emerging roles for hyaluronidase in cancer metastasis and therapy.

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past 5 years have clarified many apparent contradictions: (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high-molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature supporting these conclusions and discusses novel mechanisms by which hyaluronidases impact invasive tumor cell processes. In addition, a detailed structural and functional comparison of the hyaluronidases is presented with insights into substrate selectivity and potential for therapeutic targeting. Finally, technological advances in targeting hyaluronidase for tumor imaging and cancer therapy are summarized.

Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.

Germ-cell hyaluronidases: their roles in sperm function.

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm.

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Signaling from membrane receptors to tumor suppressor WW domain-containing oxidoreductase.

The family of WW domain-containing proteins contains over 2000 members. The small WW domain module is responsible, in part, for protein/protein binding interactions and signaling. Many of these proteins are located at the membrane/cytoskeleton area, where they act as adaptors to receive signals from the cell surface. In this review, we provide molecular insights regarding recent novel findings on signaling from the cell surface toward WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1. More specifically, transforming growth factor beta 1 utilizes cell surface hyaluronidase Hyal-2 (hyaluronoglucosaminidase 2) as a cognate receptor for signaling with WWOX and Smad4 to control gene transcription, growth and death. Complement C1q alone, bypassing the activation of classical pathway, signals a novel event of apoptosis by inducing microvillus formation and WWOX activation. Deficiency in these signaling events appears to favorably support cancer growth.

Hyaluronan-degrading activity of mouse sperm hyaluronidase is not required for fertilization?

Sperm hyaluronidase has long been believed to participate in sperm penetration through the cumulus matrix. However, our previous works using male mice lacking either one of two sperm hyaluronidases, SPAM1 and HYAL5, conclusively showed that neither of these hyaluronidases is essential for fertilization. In this study, we examined whether the hyaluronan-degrading activity of mouse epididymal sperm is indeed required for the fertilization process. When the oocyte-cumulus complex was incubated with sperm protein extracts or capacitated epididymal sperm in the presence of the hyaluronidase inhibitor apigenin, dispersal of cumulus cells from the cumulus was effectively inhibited. Despite the presence of apigenin, capacitated epididymal sperm normally entered the oocyte-cumulus complex, traversed the cumulus matrix and reached the oocyte zona pellucida. Importantly, epididymal sperm were also capable of normally fertilizing the metaphase II-arrested oocytes in the presence of apigenin. These data suggest that the hyaluronan-degrading activity of sperm hyaluronidase may not be required for fertilization, at least in the mouse.

SPAM1 isoforms from two tissue origins are differentially localized within ejaculated bull sperm membranes and have different roles during fertilization.

The deduced amino acid sequence of bull sperm, SPAM1, suggests that it possesses a transmembrane domain between the hyaluronidase and the putative zona pellucida (ZP) binding domains. The objective of this study was to determine the orientation and localization of SPAM1 in order to understand how it could fulfill these two roles. We report that two isoforms of SPAM1 are present on ejaculated bull spermatozoa: one localized on the anterior portion of the sperm head, and the other on the postacrosomal portion of the head. The first isoform is expressed intracellularly, while the second one is detected at the sperm surface with its hyaluronidase domain facing the extracellular environment. Two-dimensional electrophoresis revealed that the two isoforms have different masses (80 and 70 kDa, respectively), and LC/MS/MS analyses confirmed our previously published deduced amino acid sequence of bovine SPAM1. In addition, this approach showed that the 70-kDa isoform differs from the 80-kDa isoform in its C terminus. Our results suggest that the shorter SPAM1 form originates from the epididymis, while the longer one is produced during spermatogenesis. These results clearly demonstrate that ejaculated bull sperm possess two forms of SPAM1: one (epididymal) expressed at the surface, and one (testicular) that interacts with the ZP after the acrosome exocytosis.

Development of the pig placenta.

Placental insufficiency results in fetal loss, low birth weight, stillbirth, preweaning mortality and poor growth. Placental development begins at conceptus elongation, which is a primary factor controlling the size of the placenta. After elongation, the allantois develops outward from the embryo to establish the allantochorion, which defines the size of the functional placenta. During implantation, chorionic trophoblasts adhere to endometrial epithelial cells. Placental structures known as areolae develop at the openings of the endometrial glands and take up endometrial gland secreted products (histotrophe). Between day 30 and 35 of gestation, the adhered trophoblast-endometrial epithelial bilayer undergoes microscopic folding. Fetal and maternal capillaries develop adjacent to the bilayer and blood flows are arranged in a cross-countercurrent manner. Except for nutrients secreted by the glands, nutrient exchange takes place between these capillaries within these folds. By day 85, the folds deepen and become more complex, increasing surface area. The epithelial bilayer thins and capillaries indent the plane of each layer (but do not penetrate), reducing distance between capillaries. The folded bilayer is surrounded by endometrial stroma on the maternal side and placental stroma on the fetal side. The fetal-placental stroma is partially composed of glycosaminoglycans, the most abundant being hyaluronan and heparan sulfate. Changes in both hyaluronoglucosaminidase and heparanase during placental development suggest that these enzymes play a role in placental development. In addition to structural modifications, various nutrient specific transport mechanisms exist. These mechanisms are likely to be as important to transport of specific nutrients as placental size or structure.

Changes in neuropil ultrastructure in hippocampal field CA1 in rat pups after application of hyaluronidase.

Electron microscopy and electrophysiological studies were performed on cross-sectional slices of the hippocampus of four-week-old male rat pups (n = 35) to detect ultrastructural changes in hippocampal field CA1 and the characteristics of the formation of excitatory postsynaptic potentials in this area of the brain after incubation of slices in hyaluronidase solution (10 U/ml), whose specific substrate is the extracellular matrix glycosaminoglycan hyaluronic acid. At 1.5 min after enzyme application, there were reductions in synaptic cleft widths in axodendritic contacts of the striatum radiatum of hippocampal field CA1 by 15-25%, which were consistent with increases seen in the amplitudes of excitatory postsynaptic potentials. At 4.5 min of incubation, the lumens of synaptic clefts decreased by 45-55%: during this time there was blockade of signal transmission via Schäffer collaterals to hippocampal field CA1. Thus, the structural-functional state of glycosaminoglycans is among the factors determining the efficiency of synaptic transmission in the brain.

Hyaluronidase inhibitors: a biological and therapeutic perspective.

The hyaluronidases (HAases) are a group of less extensively studied glycosidases distributed throughout the animal kingdom and are popularly known as 'spreading factors'. In recent years, HAases received much attention due to their ability to abruptly alter the hyaluronic acid (HA) homeostasis. HAases preferentially degrade HA, which is a megadalton acidic structural polysaccharide found exclusively in the extracellular matrix (ECM) of animals. The HA-HAase system has been suggested to participate in many pathophysiological conditions. The HA degradation in ECM, crack down the structural integrity with an eventual increased tissue permeability that is attributed for the spreading property. The spreading property has been widely accepted in functions including envenomation, acrosomal reaction/ovum fertilization, cancer progression, microbial pathogenesis such as wound infections, pneumonia, and other sepses like, bacteremia and meningitis. HA fragmentation has dual effects; generation of a wide molecular range bioactive oligosaccharides of angiogenic, pro-inflammatory, and immunostimulatory properties; and impairment in the reservoir capacity of ECM that holds metal ions, growth factors, cytokines and various enzymes for signal transduction. Hence, inhibition of HA degradation appears critical and imperative in HAase mediated pathological conditions. HAase inhibitors are thus potent regulators that maintain HA homeostasis and they might serve as anti-inflammatory, anti-aging, anti-microbial, anticancer and anti-venom/toxin and contraceptive agents. In addition, HAase inhibitors may serve as tools to understand several unexplained and complex functions of HAases in HA metabolism. Therefore, this review is expected to provide an integrated update as of 2008 on the HAase inhibitors and their possible role as therapeutics in the management of a wide range of pathological conditions.

Transforming growth factor beta1 signaling via interaction with cell surface Hyal-2 and recruitment of WWOX/WOX1.

Transforming growth factor beta (TGF-beta) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-beta1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF-beta receptor-deficient HCT116 cells, as determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2.WOX1 complexes for relocation to the nuclei. TGF-beta1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast two-hybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8-9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-beta1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-beta1-induced apoptosis. Together, TGF-beta1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death.

Decreased hyaluronan in airway smooth muscle cells from patients with asthma and COPD.

Glycosaminoglycans (GAG) are essential extracellular matrix molecules which regulate tissue flexibility, a parameter that is reduced in airways of patients with asthma and chronic obstructive pulmonary disease (COPD). We investigated the expression of GAG and their metabolising enzymes in primary human airway smooth muscle cells (ASMC) obtained from healthy donors (controls) and patients with asthma or COPD. Total GAG synthesis was assessed by [(3)H]-glucosamine incorporation. GAG were isolated, purified, fractionated by electrophoresis and characterised using specific GAG-degrading enzymes. Secretion of hyaluronic acid (HA) by ASMC from patients with asthma or COPD was significantly decreased compared with controls. RT-PCR analysis and western blotting revealed that this decrease was associated with a significant reduction in the expression of HA synthase-1 and -2 and a significant increase of hyaluronidase-1. Furthermore, the expression of the HA receptor CD44 was significantly decreased, whereas the receptor for HA-mediated motility was not expressed in asthma or COPD. Our results indicate that there is a decreased expression of HA in asthma and COPD associated with a synergistic regulation of HA metabolising enzymes that may regulate the pathological airway remodelling in these lung diseases.

[Changes of the neuropil ultrastructure in the CA1 hippocampal area of rat pups after hyaluronidase treatment].

Electron microscopical and electrophysiological studies were carried out in cross-sections of the hippocampus of 4-weeks-old male rat pups (n = 35) to detect the ultrastructural changes in CA1 hippocampal area and the peculiarities of excitatory postsynaptic potential formation in this brain area after the incubation of the sections in the solution of hyaluronidase (10 U/ml), the enzyme which specifically degrades the extracellular matrix glycosaminoglycan--the hyaluronic acid. The reduction of the width of synaptic cleft by 15-25% in the axo-dendritic contacts of the stratum radiatum of CA1 hippocampal area was detected 1.5 min following the application of enzyme, coinciding with the increase of the excitatory postsynaptic potential amplitude. The width of synaptic cleft was further reduced by 45-55% after 4.5 min of incubation; during this period the blockade of signal transmission along the Schaffer's collaterals to CA1 hippocampal area was observed. Thus, the structural and functional state of glycosaminoglycans is one of the factors controlling the efficiency of synaptic transmission in the brain.

Epigenetic regulation of HYAL-1 hyaluronidase expression. identification of HYAL-1 promoter.

HYAL-1 (hyaluronoglucosaminidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 is a marker for cancer diagnosis and a molecular determinant of tumor growth, invasion, and angiogenesis. The regulation of HYAL-1 expression is unknown. Real time reverse transcription-PCR using 11 bladder and prostate cancer cells and 69 bladder tissues showed that HYAL-1 mRNA levels are elevated 10-30-fold in cells/tissues that express high hyaluronidase activity. Although multiple transcription start sites (TSS) for HYAL-1 mRNA were detected in various tissues, the major TSS in many tissues, including bladder and prostate, was at nucleotide 27274 in the cosmid clone LUCA13 (AC002455). By analyzing the 1532 base sequence 5' to this TSS, using cloning and luciferase reporter assays, we identified a TACAAA sequence at position -31 and the minimal promoter region between nucleotides -93 and -38. Mutational analysis identified that nucleotides -73 to -50 (which include overlapping binding consensus sites for SP1, Egr-1, and AP-2), bases C(-71) and C(-59), and an NFkappaB-binding site (at position -15) are necessary for promoter activity. The chromatin immunoprecipitation assay identified that Egr-1, AP-2, and NFkappaB bind to the promoter in HYAL-1-expressing cells, whereas SP1 binds to the promoter in non-HYAL-1-expressing cells. 5-Aza-2'-deoxycytidine treatment, bisulfite DNA sequencing, and methylation-specific PCR revealed that HYAL-1 expression is regulated by methylation at C(-71) and C(-59); both Cs are part of the SP1/Egr-1-binding sites. Thus, HYAL-1 expression is epigenetically regulated by the binding of different transcription factors to the methylated and unmethylated HYAL-1 promoter.