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Expression of heparanase in human tumor cell lines and human head and neck tumors
Cancer Letters 193 (2003) 83–89 www.elsevier.com/locate/canlet
Expression of heparanase in human tumor cell lines and human head and neck tumors Siro Simizua, Keisuke Ishidaa, Michal K. Wierzbaa, Taka-Aki Satob, Hiroyuki Osadaa,* a
b
Antibiotics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan College of Physicians and Surgeons, Columbia University, New York, NY 10032-3748, USA
Received 20 August 2002; received in revised form 18 November 2002; accepted 2 December 2002
Abstract Heparanase is an endo-b-D -glucuronidase that can cleave heparan sulfate and has been implicated in tumor angiogenesis and metastasis. Recent studies have demonstrated that overexpression of heparanase in human tumors facilitates their invasion activity, thereby enhancing the metastatic potential of the tumors. We found that heparanase mRNA and heparanase protein were constitutively elevated in some human tumor cell lines and human head and neck tumors. Heparanase mRNA expression was increased in response to treatment with an inhibitor of DNA methylation in cells that normally express low levels of heparanase mRNA. Inhibition of DNA methylation did not enhance heparanase mRNA expression in the presence of cycloheximide. These results suggest that overexpression of heparanase mRNA in cancer cells might not be due to demethylation of the promoter region of the heparanase gene itself, rather the other gene(s), such as transcriptional factors that, in turn, regulate heparanase expression. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Heparanase; DNA methylation; Head and neck tumor; Invasion
1. Introduction The extracellular matrix (ECM) is composed of a complex network of macromolecules that fill the extracellular space in tissues and provide a molecular scaffolding for cells within organs. Heparan sulfate proteoglycans (HSPGs) are covalently linked up with protein glycosaminoglycan conjugates found in ECM that play critical roles in cell-cell and cell-matrix interactions [1,2]. The enzymatic cleavage of heparan * Corresponding author. Tel.: þ81-48-467-9541; fax: þ 81-48462-4669. E-mail address: [email protected] (H. Osada).
sulfate chains by heparanase is critical for regulation of the biological function of heparan sulfate-binding proteins such as fibroblast growth factors [3 – 5]. The cloning of the human heparanase cDNA was independently reported by several groups [6 – 9]. The human heparanase cDNA contains an open reading frame that encodes a polypeptide of 543 amino acids with a molecular weight of 61.2 kD. Expression of the human heparanase mRNA in normal tissues is restricted primarily to the placenta and lymphoid organs [10]. Pathological increases in heparanase mRNA expression and heparanase protein levels have been reported in pancreatic cancers [11], hepatocellular carcinomas [12] and esophageal
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 1 9 - X
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carcinomas [13], however the mechanism(s) causing tumors to overexpress heparanase is still unclear. Cytosine methylation of CpG islands is an epigenetic mechanism by which genes are silenced through modulation of chromatin structure [14]. Aberrant hypermethylation of 50 CpG islands has been implicated in the transcriptional silencing of various genes, including CACNA1G [15], 14-3-3s [16], caspase-8 [17], p16 INK4a, p15 INK4b, and APC in human cancers [18]. In contrast, it has been reported that reduced methylation at the promoter region of MDR1 leads to overexpression of MDR protein in bladder cancer during chemotherapeutic treatment [19]. Thus, alterations in DNA methylation/demethylation at the promoter region may be a general mechanism for the regulation of genes that are silenced or overexpressed in various malignancies. To achieve the screening of new heparanase inhibitor, we cloned the heparanase gene and established heparanase-overexpressing cell lines. During this study, we noticed that some tumor cell lines overexpress heparanase mRNA and heparanase protein, but normal cell line does not. In this report, we investigate the expression of heparanase in some human tumor cell lines and human head and neck tumors.
2. Materials and methods 2.1. Cell culture Human cell lines, HepG2, HeLa, A431, A549 and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). Human leukemia cell lines, U937, K562, Jurkat, and HL-60 cells were cultured in RPMI medium containing 10% FBS. For the DNA demethylation assay, cells were cultured with 5-aza-20 deoxycytidine (AzaC) for 3 days, and the medium and drug were replaced every 24 h [20]. 2.2. RNA extraction and semi-quantitative PCR Total RNA was isolated from the cells using Isogen reagent following the recommendation of the manufacturer (Nippon gene). The total RNA was reverse transcribed, and polymerase chain reaction (PCR) was
performed for heparanase using the respective primer sets to amplify cDNA (total heparanase: HP1 and HP2; heparanase 1a: HP3 and HP4; heparanase 1b: HP5 and HP4). Detection of human glyceraldehyde-3phosphate dehydrogenase (GAPDH ) mRNA by semiquantitative PCR was used as a positive control to verify the efficiency of the reverse transcription (RT) reaction and the amount of RNA added to the reaction mixture [21]. The number of PCR cycles for each product was determined after confirmation of the efficacy of amplification and defining the linear exponential portion of the amplification. HP1, 50 AAAAAGTTCAAGAACAGCACC-30 ; HP2, 50 CTAGTATATTTTATTTTCAG-3 0 ; HP3, 50 0 CATCTCCGCACCCTTCAAGTG-3 ; HP4, 50 GTGCTGTTCTTGAACTTTTTC-30 ; HP5, 50 -GAGGAAGTGCTAGAGCTCTCG-30 . 2.3. Western blotting Cells were lysed in lysis buffer (10 mM HEPES, 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2% Nonidet P-40, 0.1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2) at 4 8C with sonication. The lysates were centrifuged at 14 000 rpm for 15 min. The amount of protein in each lysate was measured by staining with Coomassie Brilliant Blue G-250. Loading buffer (42 mM Tris– HCl, pH 6.8, 10% glycerol, 2.3% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.002% bromophenol blue) was then added to each lysate, and samples were electrophoresed on SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes and immunoblotted with anti-heparanase (BD Biosciences), or anti-a-tubulin (Sigma) antibodies. 2.4. Tissue samples Human head and neck tumor tissue samples were obtained from six patients undergoing surgical resection. The surrounding normal tissue was also obtained. Informed consent for the use of the sample was obtained at Columbia University. The samples were stored at 2 70 8C. Total RNA was isolated and semi-quantitative PCR was used to detect heparanase mRNA, as described before [21].
S. Simizu et al. / Cancer Letters 193 (2003) 83–89
3. Results 3.1. Heparanase mRNA and heparanase protein expressions are elevated in human tumor cell lines We analyzed heparanase mRNA expression levels by semi-quantitative PCR in several human cell lines. Among solid tumor-derived cell lines, the expression levels in HeLa, A431, and A549 cells were high, however, the level was relatively low in HepG2 cells (Fig. 1A). Heparanase mRNA in WI-38 cells, human normal lung fibroblasts, was undetectable under our assay conditions (Fig. 1A). In leukemia cell lines, a high level of expression was observed in U937 cells, whereas the expression levels in Jurkat, HL-60, and K562 cells were low (Fig. 1A). The expression level of heparanase mRNA correlated that of heparanase protein (Fig. 1B), suggesting that our semi-quantitative PCR analysis is confirmative to detect the expression levels of heparanase mRNA and heparanase protein in human tumor cell lines. It has been reported that the heparanase gene is expressed as two mRNA species, heparanase 1a mRNA and heparanase 1b mRNA, that are generated by alternative splicing and contain the same open reading frame [22]. To examine whether the high levels of heparanase mRNA expression we observed in human tumor cell lines were due to the heparanase 1a mRNA or heparanase 1b mRNA, we performed splicing form-specific semi-quantitative PCR. Heparanase 1b mRNA was found in HeLa, A431 and U937 cells, and heparanase 1a mRNA was detected in HeLa, A549 and U937 cells (Fig. 1A).
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A549, and U937), which constantly express high levels of heparanase mRNA (Fig. 2). Thus, the effect of AzaC on heparanase mRNA expression was observed only in the cells expressing low baseline levels of heparanase mRNA including normal cell line (Table 1). To determine if there were differential effects of AzaC on the expression levels of heparanase 1a mRNA and heparanase 1b mRNA, we carried out splicing form-specific semi-quantitative PCR after AzaC treatment in human tumor cell lines. Both heparanase 1a and 1b mRNAs were increased by the treatment with AzaC in the cells with low expression levels of heparanase (Fig. 2). Therefore, these results indicated that aberrant DNA demethylation may be responsible for the overexpression of heparanase mRNA in human tumor cell lines. To clarify whether the effect of AzaC on expression of heparanase mRNA was direct or indirect, we treated cells with cycloheximide, a protein synthesis inhibitor. The AzaC-induced enhancement of heparanase mRNA was inhibited in the presence of cyclohex-
3.2. Inhibition of DNA methylation induces heparanase mRNA expression in the cells with low expression levels of heparanase To determine whether DNA methylation/demethylation played a role in heparanase mRNA expression, we tested the effect of treatment with AzaC, an inhibitor of DNA methylation, on the expression of heparanase mRNA in human cell lines. As shown in Fig. 2, the expression level of heparanase mRNA was elevated in response to AzaC treatment in five cell lines (WI-38, HepG2, Jurkat, HL-60, and K562), which have relatively low levels of heparanase mRNA, but not in four cell lines (HeLa, A431,
Fig. 1. Increased expression of heparanase mRNA and heparanase protein in human tumor cell lines. (A) Relative levels of heparanase, heparanase 1a, heparanase 1b and GAPDH mRNAs were analyzed by semi-quantitative PCR using specific primers as described in materials and methods. (B) Exponentially growing cells were lysed and aliquots of cell lysates were immunoblotted with the indicated antibodies.
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Fig. 2. Induction of heparanase expression by the treatment with AzaC. Human tumor cell lines were treated with 10 mM AzaC for 3 days. The mRNAs were prepared from cells, and the relative levels of heparanase, heparanase 1a, heparanase 1b, and GAPDH mRNAs were analyzed by semi-quantitative PCR using specific primers as described in Section 2.
imide (Fig. 3), indicating that the increase in heparanase mRNA levels was dependent on de novo protein synthesis. These results suggest that overexpression of heparanase in cancer cells might not be caused by the aberrant demethylation of heparanase gene itself, rather the other gene(s), such as transcriptional factors that, in turn, regulate heparanase expression. 3.3. Expression of heparanase mRNA in human head and neck tumors Pathological increases in heparanase mRNA expression and heparanase protein levels have been
reported in pancreatic cancers, hepatocellular carcinomas and esophageal carcinomas [11 –13], however expression levels of heparanase mRNA in other tumors have not been reported. We analyzed heparanase mRNA in human head and neck tumors by semi-quantitative PCR, because head and neck tumor is a major cause of cancer morbidity and mortality worldwide with more than 500 000 new cases reported annually [23]. Up-regulation of heparanase mRNA, compared with normal tissues, was observed in tumor RNA from three out of six patients (Fig. 4). Heparanase 1a mRNA was up-regulated in two of six patients, and heparanase 1b mRNA was the variant identified in three of six patients (Fig. 4). All tumors
Table 1 Effect of AzaC treatment on expression of heparanase mRNA in human tumor cell lines Human cell line
Origin
Heparanase mRNA levela
Increase of heparanase mRNA by AzaC treatmentb
WI-38 HeLa A431 HepG2 A549 U937 Jurkat HL-60 K562
Normal lung fibroblast Cervical carcinoma Epidermoid carcinoma Hepatoma Lung adenocarcinoma Promonocytic leukemia T-cell leukemia Promyelocytic leukemia Erythroleukemia
2 þ þ 2 þ þ 2 2 2
þ 2 2 þ 2 2 þ þ þ
a Heparanase mRNA level was determined by RT-PCR as shown in Fig. 1. þ , heparanase mRNA was detected; 2, heparanase mRNA was not detected. b Increase of heparanase mRNA by AzaC treatment was assessed by RT-PCR. þ, heparanase mRNA was increased after AzaC treatment; 2, heparanase mRNA was not changed after AzaC treatment.
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Fig. 3. Regulation of AzaC-induced heparanase expression by de novo protein synthesis. HepG2 cells were pretreated with 10 mg/ml cycloheximide for 1 h, and treated with 100 mM AzaC for 1 day. Heparanase and GAPDH mRNAs were then analyzed by semiquantitative PCR.
exhibiting elevated heparanase mRNA expressed heparanase 1b mRNA, and two of these tumors also expressed heparanase 1a mRNA.
4. Discussion Invasion and secondary spread through the blood and lymphatic system are characteristic features of malignant tumors. This process represents one of the greatest impediments to curing cancer. Many mechanisms are involved in this complex process, and cell adhesion molecules, such as cadherin [24] and cell surface HSPGs are especially important factors in the regulation of cell differentiation, morphology and migration [1 – 5,25]. Heparanase is an endoglucuronidase that specifically degrades heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. Expression of the human heparanase mRNA in normal tissues is observed primarily in the placenta and lymphoid organs [10]. High levels of heparanase mRNA expression are also observed in many human tumors [11 –13]. In the present study, we found high levels of heparanase mRNA and heparanase protein expression in several human tumor cell lines, including HeLa, A431, A549 and U937 cells (Fig. 1). It has been shown that the heparanase gene is expressed as two mRNA species containing the same open reading frame, heparanase 1a mRNA and heparanase 1b mRNA, generated by alternative splicing [22]. Heparanase 1a mRNA is the major transcript of the heparanase gene in the human immune system (including spleen and peripheral blood leukocytes), whereas heparanase 1b mRNA is
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the major transcript in human placenta, platelets and transformed-mouse fibroblast cells [22]. In this study we observed both heparanase 1a and heparanase 1b mRNA species in tumor cells and tumor-derived cell lines, suggesting that both mRNA species may be responsible for tumor cell invasion. Further studies are necessary to determine the mechanism underlying the separate regulation of heparanase 1a and heparanase 1b mRNA expression. We found that five cell lines (WI-38, HepG2, Jurkat, HL-60, and K562) express only a trace amount of heparanase mRNA in the absence of AzaC, and that AzaC treatment induced the expression of heparanase mRNA via de novo protein synthesis in these cell lines (Figs. 2 and 3). On the other hand, AzaC treatment did not affect the levels of heparanase mRNA in cells, such as HeLa cells, with high baseline levels of heparanase mRNA expression (Table 1). Our results suggest that the low levels of heparanase mRNA expression in most normal human tissues may be maintained by the DNA methylation and that aberrant demethylation of the gene(s), unlikely heparanase gene promoter region itself, might occur in the heparanase mRNA expression pathways in human tumors and tumor-derived cell lines. Head and neck tumor is characterized by local tumor aggressiveness and a marked propensity for dissemination to cervical lymph nodes [23]. Whereas the management of head and neck tumor has improved, there is no evidence to suggest that therapeutic advances have resulted in increased
Fig. 4. Increased expression of heparanase mRNA in human head and neck tumors. Relative levels of heparanase, heparanase 1a, heparanase 1b and GAPDH mRNAs were analyzed by semiquantitative PCR using specific primers as described in Section 2. Normal and tumor tissues are designated N and T, respectively.
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survival rates. We demonstrated that overexpression of heparanase mRNA was observed in human head and neck tumors (Fig. 4). However, overexpression of heparanase mRNA was observed in not all the tumors. Thus, the regulation of heparanase expression levels represents a critical determinant of whether head and neck tumors actively invade or not invade. Taken together, our data reveal that pathological increase in heparanase mRNA expression is regulated by the several mechanisms, such as aberrant DNA demethylation. This study represents the first evidence concerning the mechanisms underlying regulation of heparanase mRNA expression in human tumor cells. Therefore, drugs that can inhibit the level or activity of heparanase protein might be useful for cancer chemotherapy.
[8]
[9]
[10]
[11]
Acknowledgements [12]
The authors thank M. Toyota (Sapporo Medical University) for helpful suggestions and discussions and Y. Ichikawa and R. Nakazawa for DNA sequencing (Bioarchitect Research Group, RIKEN). This study was partly supported by a Grant-in Aid from the Ministry of Education, Science, Sports, Culture, and Technology of Japan, and by the Multibioprobe project (RIKEN).
[13]
[14]
[15]
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[23] E.E. Vokes, R.R. Weichselbaum, S.M. Lippman, W.K. Hong, Head and neck cancer, N. Engl. J. Med. 328 (1993) 184–194. [24] M. Takeichi, Cadherin cell adhesion receptors as a morphogenetic regulator, Science 251 (1991) 1451–1455. [25] K.J. Bame, Heparanase: endoglycosidases that degrade heparan sulfate proteoglycans, Glycobiology 11 (2001) 91R–98R.
Expression of heparanase in human tumor cell lines and human head and neck tumors Siro Simizua, Keisuke Ishidaa, Michal K. Wierzbaa, Taka-Aki Satob, Hiroyuki Osadaa,* a
b
Antibiotics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan College of Physicians and Surgeons, Columbia University, New York, NY 10032-3748, USA
Received 20 August 2002; received in revised form 18 November 2002; accepted 2 December 2002
Abstract Heparanase is an endo-b-D -glucuronidase that can cleave heparan sulfate and has been implicated in tumor angiogenesis and metastasis. Recent studies have demonstrated that overexpression of heparanase in human tumors facilitates their invasion activity, thereby enhancing the metastatic potential of the tumors. We found that heparanase mRNA and heparanase protein were constitutively elevated in some human tumor cell lines and human head and neck tumors. Heparanase mRNA expression was increased in response to treatment with an inhibitor of DNA methylation in cells that normally express low levels of heparanase mRNA. Inhibition of DNA methylation did not enhance heparanase mRNA expression in the presence of cycloheximide. These results suggest that overexpression of heparanase mRNA in cancer cells might not be due to demethylation of the promoter region of the heparanase gene itself, rather the other gene(s), such as transcriptional factors that, in turn, regulate heparanase expression. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Heparanase; DNA methylation; Head and neck tumor; Invasion
1. Introduction The extracellular matrix (ECM) is composed of a complex network of macromolecules that fill the extracellular space in tissues and provide a molecular scaffolding for cells within organs. Heparan sulfate proteoglycans (HSPGs) are covalently linked up with protein glycosaminoglycan conjugates found in ECM that play critical roles in cell-cell and cell-matrix interactions [1,2]. The enzymatic cleavage of heparan * Corresponding author. Tel.: þ81-48-467-9541; fax: þ 81-48462-4669. E-mail address: [email protected] (H. Osada).
sulfate chains by heparanase is critical for regulation of the biological function of heparan sulfate-binding proteins such as fibroblast growth factors [3 – 5]. The cloning of the human heparanase cDNA was independently reported by several groups [6 – 9]. The human heparanase cDNA contains an open reading frame that encodes a polypeptide of 543 amino acids with a molecular weight of 61.2 kD. Expression of the human heparanase mRNA in normal tissues is restricted primarily to the placenta and lymphoid organs [10]. Pathological increases in heparanase mRNA expression and heparanase protein levels have been reported in pancreatic cancers [11], hepatocellular carcinomas [12] and esophageal
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 1 9 - X
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carcinomas [13], however the mechanism(s) causing tumors to overexpress heparanase is still unclear. Cytosine methylation of CpG islands is an epigenetic mechanism by which genes are silenced through modulation of chromatin structure [14]. Aberrant hypermethylation of 50 CpG islands has been implicated in the transcriptional silencing of various genes, including CACNA1G [15], 14-3-3s [16], caspase-8 [17], p16 INK4a, p15 INK4b, and APC in human cancers [18]. In contrast, it has been reported that reduced methylation at the promoter region of MDR1 leads to overexpression of MDR protein in bladder cancer during chemotherapeutic treatment [19]. Thus, alterations in DNA methylation/demethylation at the promoter region may be a general mechanism for the regulation of genes that are silenced or overexpressed in various malignancies. To achieve the screening of new heparanase inhibitor, we cloned the heparanase gene and established heparanase-overexpressing cell lines. During this study, we noticed that some tumor cell lines overexpress heparanase mRNA and heparanase protein, but normal cell line does not. In this report, we investigate the expression of heparanase in some human tumor cell lines and human head and neck tumors.
2. Materials and methods 2.1. Cell culture Human cell lines, HepG2, HeLa, A431, A549 and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). Human leukemia cell lines, U937, K562, Jurkat, and HL-60 cells were cultured in RPMI medium containing 10% FBS. For the DNA demethylation assay, cells were cultured with 5-aza-20 deoxycytidine (AzaC) for 3 days, and the medium and drug were replaced every 24 h [20]. 2.2. RNA extraction and semi-quantitative PCR Total RNA was isolated from the cells using Isogen reagent following the recommendation of the manufacturer (Nippon gene). The total RNA was reverse transcribed, and polymerase chain reaction (PCR) was
performed for heparanase using the respective primer sets to amplify cDNA (total heparanase: HP1 and HP2; heparanase 1a: HP3 and HP4; heparanase 1b: HP5 and HP4). Detection of human glyceraldehyde-3phosphate dehydrogenase (GAPDH ) mRNA by semiquantitative PCR was used as a positive control to verify the efficiency of the reverse transcription (RT) reaction and the amount of RNA added to the reaction mixture [21]. The number of PCR cycles for each product was determined after confirmation of the efficacy of amplification and defining the linear exponential portion of the amplification. HP1, 50 AAAAAGTTCAAGAACAGCACC-30 ; HP2, 50 CTAGTATATTTTATTTTCAG-3 0 ; HP3, 50 0 CATCTCCGCACCCTTCAAGTG-3 ; HP4, 50 GTGCTGTTCTTGAACTTTTTC-30 ; HP5, 50 -GAGGAAGTGCTAGAGCTCTCG-30 . 2.3. Western blotting Cells were lysed in lysis buffer (10 mM HEPES, 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2% Nonidet P-40, 0.1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2) at 4 8C with sonication. The lysates were centrifuged at 14 000 rpm for 15 min. The amount of protein in each lysate was measured by staining with Coomassie Brilliant Blue G-250. Loading buffer (42 mM Tris– HCl, pH 6.8, 10% glycerol, 2.3% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.002% bromophenol blue) was then added to each lysate, and samples were electrophoresed on SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes and immunoblotted with anti-heparanase (BD Biosciences), or anti-a-tubulin (Sigma) antibodies. 2.4. Tissue samples Human head and neck tumor tissue samples were obtained from six patients undergoing surgical resection. The surrounding normal tissue was also obtained. Informed consent for the use of the sample was obtained at Columbia University. The samples were stored at 2 70 8C. Total RNA was isolated and semi-quantitative PCR was used to detect heparanase mRNA, as described before [21].
S. Simizu et al. / Cancer Letters 193 (2003) 83–89
3. Results 3.1. Heparanase mRNA and heparanase protein expressions are elevated in human tumor cell lines We analyzed heparanase mRNA expression levels by semi-quantitative PCR in several human cell lines. Among solid tumor-derived cell lines, the expression levels in HeLa, A431, and A549 cells were high, however, the level was relatively low in HepG2 cells (Fig. 1A). Heparanase mRNA in WI-38 cells, human normal lung fibroblasts, was undetectable under our assay conditions (Fig. 1A). In leukemia cell lines, a high level of expression was observed in U937 cells, whereas the expression levels in Jurkat, HL-60, and K562 cells were low (Fig. 1A). The expression level of heparanase mRNA correlated that of heparanase protein (Fig. 1B), suggesting that our semi-quantitative PCR analysis is confirmative to detect the expression levels of heparanase mRNA and heparanase protein in human tumor cell lines. It has been reported that the heparanase gene is expressed as two mRNA species, heparanase 1a mRNA and heparanase 1b mRNA, that are generated by alternative splicing and contain the same open reading frame [22]. To examine whether the high levels of heparanase mRNA expression we observed in human tumor cell lines were due to the heparanase 1a mRNA or heparanase 1b mRNA, we performed splicing form-specific semi-quantitative PCR. Heparanase 1b mRNA was found in HeLa, A431 and U937 cells, and heparanase 1a mRNA was detected in HeLa, A549 and U937 cells (Fig. 1A).
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A549, and U937), which constantly express high levels of heparanase mRNA (Fig. 2). Thus, the effect of AzaC on heparanase mRNA expression was observed only in the cells expressing low baseline levels of heparanase mRNA including normal cell line (Table 1). To determine if there were differential effects of AzaC on the expression levels of heparanase 1a mRNA and heparanase 1b mRNA, we carried out splicing form-specific semi-quantitative PCR after AzaC treatment in human tumor cell lines. Both heparanase 1a and 1b mRNAs were increased by the treatment with AzaC in the cells with low expression levels of heparanase (Fig. 2). Therefore, these results indicated that aberrant DNA demethylation may be responsible for the overexpression of heparanase mRNA in human tumor cell lines. To clarify whether the effect of AzaC on expression of heparanase mRNA was direct or indirect, we treated cells with cycloheximide, a protein synthesis inhibitor. The AzaC-induced enhancement of heparanase mRNA was inhibited in the presence of cyclohex-
3.2. Inhibition of DNA methylation induces heparanase mRNA expression in the cells with low expression levels of heparanase To determine whether DNA methylation/demethylation played a role in heparanase mRNA expression, we tested the effect of treatment with AzaC, an inhibitor of DNA methylation, on the expression of heparanase mRNA in human cell lines. As shown in Fig. 2, the expression level of heparanase mRNA was elevated in response to AzaC treatment in five cell lines (WI-38, HepG2, Jurkat, HL-60, and K562), which have relatively low levels of heparanase mRNA, but not in four cell lines (HeLa, A431,
Fig. 1. Increased expression of heparanase mRNA and heparanase protein in human tumor cell lines. (A) Relative levels of heparanase, heparanase 1a, heparanase 1b and GAPDH mRNAs were analyzed by semi-quantitative PCR using specific primers as described in materials and methods. (B) Exponentially growing cells were lysed and aliquots of cell lysates were immunoblotted with the indicated antibodies.
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Fig. 2. Induction of heparanase expression by the treatment with AzaC. Human tumor cell lines were treated with 10 mM AzaC for 3 days. The mRNAs were prepared from cells, and the relative levels of heparanase, heparanase 1a, heparanase 1b, and GAPDH mRNAs were analyzed by semi-quantitative PCR using specific primers as described in Section 2.
imide (Fig. 3), indicating that the increase in heparanase mRNA levels was dependent on de novo protein synthesis. These results suggest that overexpression of heparanase in cancer cells might not be caused by the aberrant demethylation of heparanase gene itself, rather the other gene(s), such as transcriptional factors that, in turn, regulate heparanase expression. 3.3. Expression of heparanase mRNA in human head and neck tumors Pathological increases in heparanase mRNA expression and heparanase protein levels have been
reported in pancreatic cancers, hepatocellular carcinomas and esophageal carcinomas [11 –13], however expression levels of heparanase mRNA in other tumors have not been reported. We analyzed heparanase mRNA in human head and neck tumors by semi-quantitative PCR, because head and neck tumor is a major cause of cancer morbidity and mortality worldwide with more than 500 000 new cases reported annually [23]. Up-regulation of heparanase mRNA, compared with normal tissues, was observed in tumor RNA from three out of six patients (Fig. 4). Heparanase 1a mRNA was up-regulated in two of six patients, and heparanase 1b mRNA was the variant identified in three of six patients (Fig. 4). All tumors
Table 1 Effect of AzaC treatment on expression of heparanase mRNA in human tumor cell lines Human cell line
Origin
Heparanase mRNA levela
Increase of heparanase mRNA by AzaC treatmentb
WI-38 HeLa A431 HepG2 A549 U937 Jurkat HL-60 K562
Normal lung fibroblast Cervical carcinoma Epidermoid carcinoma Hepatoma Lung adenocarcinoma Promonocytic leukemia T-cell leukemia Promyelocytic leukemia Erythroleukemia
2 þ þ 2 þ þ 2 2 2
þ 2 2 þ 2 2 þ þ þ
a Heparanase mRNA level was determined by RT-PCR as shown in Fig. 1. þ , heparanase mRNA was detected; 2, heparanase mRNA was not detected. b Increase of heparanase mRNA by AzaC treatment was assessed by RT-PCR. þ, heparanase mRNA was increased after AzaC treatment; 2, heparanase mRNA was not changed after AzaC treatment.
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Fig. 3. Regulation of AzaC-induced heparanase expression by de novo protein synthesis. HepG2 cells were pretreated with 10 mg/ml cycloheximide for 1 h, and treated with 100 mM AzaC for 1 day. Heparanase and GAPDH mRNAs were then analyzed by semiquantitative PCR.
exhibiting elevated heparanase mRNA expressed heparanase 1b mRNA, and two of these tumors also expressed heparanase 1a mRNA.
4. Discussion Invasion and secondary spread through the blood and lymphatic system are characteristic features of malignant tumors. This process represents one of the greatest impediments to curing cancer. Many mechanisms are involved in this complex process, and cell adhesion molecules, such as cadherin [24] and cell surface HSPGs are especially important factors in the regulation of cell differentiation, morphology and migration [1 – 5,25]. Heparanase is an endoglucuronidase that specifically degrades heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. Expression of the human heparanase mRNA in normal tissues is observed primarily in the placenta and lymphoid organs [10]. High levels of heparanase mRNA expression are also observed in many human tumors [11 –13]. In the present study, we found high levels of heparanase mRNA and heparanase protein expression in several human tumor cell lines, including HeLa, A431, A549 and U937 cells (Fig. 1). It has been shown that the heparanase gene is expressed as two mRNA species containing the same open reading frame, heparanase 1a mRNA and heparanase 1b mRNA, generated by alternative splicing [22]. Heparanase 1a mRNA is the major transcript of the heparanase gene in the human immune system (including spleen and peripheral blood leukocytes), whereas heparanase 1b mRNA is
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the major transcript in human placenta, platelets and transformed-mouse fibroblast cells [22]. In this study we observed both heparanase 1a and heparanase 1b mRNA species in tumor cells and tumor-derived cell lines, suggesting that both mRNA species may be responsible for tumor cell invasion. Further studies are necessary to determine the mechanism underlying the separate regulation of heparanase 1a and heparanase 1b mRNA expression. We found that five cell lines (WI-38, HepG2, Jurkat, HL-60, and K562) express only a trace amount of heparanase mRNA in the absence of AzaC, and that AzaC treatment induced the expression of heparanase mRNA via de novo protein synthesis in these cell lines (Figs. 2 and 3). On the other hand, AzaC treatment did not affect the levels of heparanase mRNA in cells, such as HeLa cells, with high baseline levels of heparanase mRNA expression (Table 1). Our results suggest that the low levels of heparanase mRNA expression in most normal human tissues may be maintained by the DNA methylation and that aberrant demethylation of the gene(s), unlikely heparanase gene promoter region itself, might occur in the heparanase mRNA expression pathways in human tumors and tumor-derived cell lines. Head and neck tumor is characterized by local tumor aggressiveness and a marked propensity for dissemination to cervical lymph nodes [23]. Whereas the management of head and neck tumor has improved, there is no evidence to suggest that therapeutic advances have resulted in increased
Fig. 4. Increased expression of heparanase mRNA in human head and neck tumors. Relative levels of heparanase, heparanase 1a, heparanase 1b and GAPDH mRNAs were analyzed by semiquantitative PCR using specific primers as described in Section 2. Normal and tumor tissues are designated N and T, respectively.
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survival rates. We demonstrated that overexpression of heparanase mRNA was observed in human head and neck tumors (Fig. 4). However, overexpression of heparanase mRNA was observed in not all the tumors. Thus, the regulation of heparanase expression levels represents a critical determinant of whether head and neck tumors actively invade or not invade. Taken together, our data reveal that pathological increase in heparanase mRNA expression is regulated by the several mechanisms, such as aberrant DNA demethylation. This study represents the first evidence concerning the mechanisms underlying regulation of heparanase mRNA expression in human tumor cells. Therefore, drugs that can inhibit the level or activity of heparanase protein might be useful for cancer chemotherapy.
[8]
[9]
[10]
[11]
Acknowledgements [12]
The authors thank M. Toyota (Sapporo Medical University) for helpful suggestions and discussions and Y. Ichikawa and R. Nakazawa for DNA sequencing (Bioarchitect Research Group, RIKEN). This study was partly supported by a Grant-in Aid from the Ministry of Education, Science, Sports, Culture, and Technology of Japan, and by the Multibioprobe project (RIKEN).
[13]
[14]
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