Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Participation of host cells: resistance or collaboration

Matrix metalloproteinases play an important regulatory role in tissue morphogenesis, cell differentiation and motility, and tumor cell invasiveness. We have recently demonstrated elevated activity of the 92 kDa type IV collagenase (MMP-9) in human glioblastoma and in the present study examine the relative amounts of MMP-9 protein and mRNA in human gliomas and as well as the distribution of MMP-9 in human glioma tumors in vivo. Using an enzyme-linked immunosorbent assay for the quantitative determination of MMP-9 protein, we found that levels were significantly higher in malignant astrocytomas, especially in glioblastoma multiforme, than in normal brain tissues and low-grade gliomas. In addition, the amount of MMP-9 mRNA, as determined by northern blot analysis was higher in anaplastic astrocytomas and glioblastoma multiforme than in normal brain tissue and low-grade gliomas. Immunocytochemical staining for MMP-9 showed strong cytoplasmic immunoreactivity in the tumor cells and the proliferating endothelial cells of glioblastoma multiforme and anaplastic astrocytomas. The staining intensity was lower in low-grade astrocytomas, and was undetectable or very low in normal brain astrocytes. The results indicate that expression of MMP-9 is dramatically upregulated in highly malignant gliomas and correlates with the highly malignant progression of human gliomas in vivo, and support a role for the MMP-9 in facilitating the invasiveness seen in malignant gliomas in vivo.     

Expression of entry receptor nectin-1 of herpes simplex virus 1 and/or herpes simplex virus 2 in normal and neoplastic human nervous system tissues

Herpes simplex virus 1 and/or Herpes simplex virus 2 (HSV) are important pathogens of human nervous system (NS) and genetically modified HSV strains have been proposed as vectors for gene therapy targeting the brain and brain tumors. Nectin-1 is an immunoglobulin-like adhesion molecule that participates in the formation of synapses and serves as an entry receptor for HSV. The expression pattern of nectin-1 in normal human NS and brain tumors is not well understood. To better understand the nectin-1 expression in normal and neoplastic human NS, immunohistochemistry was used to detect the nectin-1 expression in sections of normal human brain, spinal cord and trigeminal and dorsal root ganglia (n=10) and in sections of primary NS neoplasms (n=22). In normal human NS, nectin-1 was detected in the soma and processes of central and peripheral neurons, in ependymal cells, choroid plexus epithelial cells, vascular endothelial cells and meningothelial cells. Oligodendrocytes, astrocytes, vascular smooth muscle cells, and Schwann cells showed variable immunoreactivity. Among tumors, schwannoma, fibrous meningioma, and medulloblastoma were nectin-1 negative. Oligodendroglioma, ependymoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme and meningothelial meningioma showed weak focal nectin-1-positivity. Ganglion cells of ganglioglioma were strongly positive. These studies provide novel information about the expression of nectin-1 in normal and neoplastic NS, and thus may lead to a better understanding of cell targeting by HSV during HSV-induced neurological disease and during a HSV-based gene therapy.     

Expression of cysteine protease inhibitors in human gliomas and meningiomas

Increased levels of human cysteine proteases have been implicated in the progression of tumors from the premalignant to the malignant state. The physiological activities of these proteases are regulated by their interactions with specific inhibitors. To our knowledge there have been no previous reports about the cysteine protease inhibitors (CPIs) in human brain tumors. In the study reported here, we determined CPI activity during glioma progression and compared that with normal human brain tissue. We also determined CPI activities in meningioma and glioblastoma cell lines in vitro. This activity was significantly higher in normal brain tissue and low-grade glioma than in anaplastic astrocytoma and glioblastoma. CPI activity was significantly higher in benign and atypical meningioma cell extracts in comparison with those from malignant meningiomas and with those from glioblastoma cell lines. After several passages, one benign meningioma cell line showed reduced levels of CPI and increased levels of cathepsin. Our results suggest that decreases in the activities of CPI may contribute to the malignant properties of brain tumors.     

钠氢交换体1在星形细胞瘤中的表达及其与恶性程度的关系*

目的：检测人星形细胞瘤和正常脑组织中钠氢交换体1（ NHE1）的表达差异及其与恶性程度的关系,探 讨星形细胞瘤增殖、生长的分子机制。方法：收集人星形细胞瘤标本51 例,低、高级别星形细胞瘤组织分别为 22 例、29 例,以肿瘤周围相对正常脑组织作为对照。用H-E 染色进行诊断和分级,免疫组织化学和免疫印迹检 测肿瘤组织与正常脑组织中NHE1表达变化。结果：NHE1主要分布在对照组神经元和少量星形胶质细胞胞膜上; 在肿瘤组织中,NHE1分布在低级别星形细胞瘤细胞膜上,并强烈表达于高级别星形细胞瘤的胞质和胞膜上。与 对照组相比较,在低级别和高级别星形细胞瘤组织中NHE1表达上调,其中,恶性程度较高的高级别肿瘤相对于 恶性程度低的低级别肿瘤,NHE1的表达更为强烈。结论：NHE1在人星形细胞瘤组织中表达增强,其强度变化 与肿瘤的恶性程度有关。     

MMP-7、MMP-10和TIMP-4在心力衰竭心室重构中的表达

目的:应用细胞因子抗体芯片技术筛选与心力衰竭心室重构关系密切的基质蛋白酶。方法:从本院的心脏病组织库中挑选6例病理诊断明确和各方面资料比较齐全的致心律失常性右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行细胞因子抗体芯片分析,筛选在致心律失常性右室心肌病引起的心力衰竭中差异表达的基质蛋白酶,并应用酶联免疫分析和免疫组织化学的方法加以验证。结果:在所筛选的17种基质金属蛋白酶中,只有MMP-7和MMP-10在致心律失常性右室心肌病引起心力衰竭中高表达,而在4种基质金属蛋白酶内源性组织抑制剂中,只有TIMP-4 低表达。经酶联免疫分析和免疫组织化学的方法证实,不仅在致心律失常性右室心肌病引起心力衰竭的心肌,在缺血性心肌病和扩张性心肌病引起的心力衰竭心肌中也发现MMP-7和MMP-10 的高表达及TIMP-4的低表达。结论:心肌组织中MMP-7和MMP-10的高表达及TIMP-4的低表达在不同心肌病引起的心力衰竭心室重构分子机制中可能发挥重要的作用。     

Expression of urokinase-type plasminogen activator receptor (uPAR) in primary central nervous system neoplasms.

The cellular receptor for urokinase-type plasminogen activator receptor (uPAR) is a member of the glycosylphosphatidylinositol (GPI) anchored protein family. It is a specific cell surface receptor for its ligand, urokinase-type plasminogen activator, which catalyzes the formation of plasmin from plasminogen to generate the proteolytic cascade and leads to the breakdown of the extracellular matrix. uPAR has been shown to correlate with a propensity to tumor invasion and metastasis in several types of non-central nervous system tumors. In this study, the authors examined the immunohistochemical expression of uPAR in 65 primary brain tumors (5 pilocytic astrocytomas, 5 diffuse astrocytomas, 6 anaplastic astrocytomas, 8 glioblastomas, 5 oligodendrogliomas, 4 oligoastrocytomas, 6 anaplastic oligoastrocytomas, 4 gangliogliomas, 4 ependymomas, 5 medulloblastomas, 6 schwannomas, 5 meningiomas, 2 atypical meningiomas). The specimens were evaluated for intensity of immunostaining (0-3 scale), cellular localization of staining, and specific or unique patterns of staining. Some degree of uPAR expression was observed in all tumors. A significant positive correlation (P = 0.0006) between tumor grade and staining intensity was identified within the astrocytoma/glioblastoma subgroup, suggesting a possible correlation with anaplastic change and propensity to tumor invasion. Expression of uPAR in nonmalignant, noninvasive tumors such as schwannoma and meningioma suggests that uPAR may have other biologic functions in addition to promotion of tumor invasion.     

Expression of eukaryotic initiation factor 4E in astrocytic tumors.

Upregulation of expression of the eukaryotic initiation factor 4E (eIF4E) has been identified in breast carcinomas, squamous cell carcinomas in the head and neck regions, and transitional cell carcinomas of urinary bladder. In this immunohistochemical study, eIF4E protein expression was investigated in human brain tissue from patients without central nervous system diseases and brain biopsy tissues from patients with anaplastic astrocytoma and glioblastoma multiforme. Expression of eIF4E protein was observed in normal pyramidal neurons but not in neuroglial components. In anaplastic astrocytoma and glioblastoma multiforme, there was diffuse uniform expression of eIF4E immunoreactivity in malignant astrocytes. A similar pattern of immunoreactivity was also present in proliferative endothelial cells and vascular lining endothelial cells in glioblastoma multiforme. This study provides evidence that eIF4E is upregulated in high-grade astrocytic tumors. As in other malignancies, a high level of eIF4E may play an important role in the neoplastic transformation, angiogenesis, and tumor growth in astrocytic tumors. Because eIF4E is crucial in regulation of tumor growth, eIF4E could be a potential target for inhibitors as an adjuvant therapy for brain tumors.     

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3-13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3-13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.     

LDH ISOZYME ANALYSES OF ETHYLNITROSOUREA-INDUCED CENTRAL NERVOUS SYSTEM TUMORS IN RATS

To provide the significance of LDH isozymes in rat CNS tumors, the changes in lactic dehydrogenase isozyme and calculated ratios of H- to M- subunit were studied by means of polyacrylamide gel enzymoelectrophoresis in tumor extracts from GNS tumors (7 astrocytomas, 4 oligodendrogliomas, 7 mixed gliomas, 6 anaplastic gliomas, 3 glioependymomas, 1 astroblastoma, 11 neurinomas, 8 anaplastic neurinomas and 1 meningioma in Wistar rats which were induced by ethylnitrosourea). The isozyme patterns were compared to those obtained from normal rat CNS tissues. Among the glioma group, oligodendroglioma showed the highest H/M ratio followed by mixed glioma, glioependymoma, astrocytoma, astroblastoma and anaplastic glioma in order of decreasing of H/M ratios. On the other hand, the H/M ratio of neurinoma was significantly higher than that of anaplastic neurinoma. These observation suggested that determination of LDH isozyme patterns could supplement the histological evaluation of brain tumors.     

人脑星形细胞瘤纤溶酶原激活抑制因子1基因表达研究

目的研究人脑星形细胞瘤纤溶酶原激活抑制因子１（ＰＡＩ１）基因表达及其临床意义。方法采用Ｎｏｒｔｈｅｒｎ杂交和免疫组化ＡＢＣ方法检测３６例人脑星形细胞瘤ＰＡＩ１ｍＲＮＡ和蛋白表达，分析其与临床病理因素之间的关系。结果所有星形细胞瘤组织均可表达３．０ｋｂ和２．２ｋｂ的ＰＡＩ１ｍＲＮＡ转录物；高分级星形细胞瘤ＰＡＩ１ｍＲＮＡ表达水平显著高于低分级星形细胞瘤（Ｐ＜００１）；正常脑组织未检测出ＰＡＩ１ｍＲＮＡ表达。ＰＡＩ１ｍＲＮＡ表达水平与星形细胞瘤的坏死（ｒ＝０．５１，Ｐ＜０．０１）、微血管数（ｒ＝０．３３，Ｐ＜０．０１）及脑水肿（ｒ＝０．２７，Ｐ＜０．０１）呈显著正相关，与患者性别、年龄及瘤体大小无显著相关性。免疫组化染色显示，ＰＡＩ１蛋白主要分布在高分级星形细胞瘤的瘤细胞和内皮细胞，尤以血管增殖部位和坏死灶周围较为显著，低分级星形细胞瘤呈低水平表达。结论ＰＡＩ１基因表达与人脑星形细胞瘤的分级、坏死、血管生成及脑水肿密切相关，可作为星形细胞瘤恶性程度的分子标记。     

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 × 10⁻⁶M and 10⁻⁵M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes. This revised version was published online in July 2006 with corrections to the Cover Date.     

High expression of tissue inhibitor of metalloproteinase-2 in serous ovarian carcinomas and the role of this expression in ovarian tumorigenesis

Tissue inhibitors of metalloproteinases (TIMPs) play key roles in maintaining homeostasis of the extracellular matrix by controlling matrix metalloproteinases (MMPs). In addition to their role in regulating MMPs, TIMPs have also been shown to have pluripotential effects on cell growth, apoptosis, and differentiation. The aim of this study was to evaluate TIMP-2 level in serous ovarian tumor tissues and to understand further the role of TIMP-2 protein in ovarian tumorigenesis. The expression of TIMP-2 was assessed by immunohistochemistry in a total of 57 ovarian specimens, including 5 normal ovaries, 12 benign serous cystadenomas, 20 serous borderline tumors, and 20 serous carcinomas. In addition, we transfected a TIMP-2 plasmid into the gynecologic cancer cell lines SKOV-3, 2774, and HeLa and then assayed cell growth, apoptosis, and MMP-2 activation. We found that TIMP-2 immunostaining was significantly more frequent in serous carcinomas, mainly in tumor epithelium, compared with cells of the other tissues studied. Tissue inhibitor of metalloproteinase-2 overexpression in ovarian cancer cells did not mediate proapoptosis, inhibited cisplatin-induced apoptosis, and induced MMP-2 expression. These findings suggest that TIMP-2 may function to favor tumor growth in serous ovarian tumorigenesis. Additional research is now needed to elucidate further the role of TIMP-2 in the biologic behavior of ovarian serous tumors.