行政院國家科學委員會專題研究計畫 期中進度報告
肺癌醣化酵素基因異常調控之研究(1/3)
計畫類別： 個別型計畫 計畫編號： NSC91-2314-B-002-268-執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立臺灣大學醫學院內科 計畫主持人： 余忠仁 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中
華
民
國 92 年 7 月 9 日
行政院國家科學委員會專題研究計畫期中報告
肺癌醣化酵素基因異常調控之研究
Dysregulation of Glycosyltransferase Genes in Lung Cancer
計畫編號：NSC91-2314-B-002-268
執行期限：91 年 8 月 1 日至 92 年 7 月 31 日
主持人：余忠仁 國立台灣大學醫學院內科
一、中文摘要 黏液素被認為與癌細胞之局部侵犯與遠隔轉移有關，由於癌細胞之黏液素基 因表現及其醣化過程異於正常細胞，有利其脫離局部病灶，並逃避宿主之免疫系 統攻擊。在吾人先前之研究中,已證實肺癌會發生黏液素基因之變異，而此變異 與異常醣化過程（涎黏液素之產生）均與肺癌病患術後之預後有關。同時並發現 涎黏液素之發生與致癌基因 neu/ erbB-2 有明顯相關性。吾人先前利用 multiplex PCR 探討肺癌之醣化酵素基因表現（包括 sialyltransferase，STs；與fucosyltransferase，FucTs），侵犯力高易轉移的細胞株（CL1-5）其 STs 與 FucTs
之表現量高於侵犯力弱的母細胞株（CL1-0、CL1-1）。臨床上，超過 50%肺癌檢體
有 STs 與 FucTs 表現量增加之現象，而表現高量 ST3GalIII 與數種 FucTs
（FucTIII、IV、VI、VII）之肺癌患者其臨床預後明顯較差，術後復發高與長期 存活率低。本三年研究計劃之目的為：(1)廣泛而完整的探討醣化酵素基因 （core1-4GnTs、β-1-4GalT I~VI、GlcNAcT I-VI）在肺癌之表現情形以及與肺癌 預後及臨床表現之相關聯性；(2)對於由(1)所選出具臨床重要性之醣化酵素基 因，探討基因表現之調控機轉。於第一年研究中，我們探討 N-acetylglucosaminyl-transferase (MGAT) I~V 在肺癌之表現量與臨床特徵及預後之相關性。
關鍵詞：肺癌、醣化酵素基因
Abstract
Mucins and mucin associated antigens are believed to play an important role in both invasion and metastasis of cancer cells. The altered expression of mucin peptides in cancer cells, as well as glycosylation, facilitates tumor cell invasion into blood stream, attachment to endothelial cells and escape from host immuno-surveillance.
Our previous studies had demonstrated the prognostic implication of mucin expression for lung cancer. Lung cancers overexpressing sialomucins tend to have higher chance of recurrence and metastasis. The expression of sialomucin is correlated with the overexpression of an oncoprotein, erbB-2/neu, and at least one mucin gene (MUC5AC apomucin). We are than interested about the altered expression of glycosyltransferase genes in lung cancer. Preliminarily, using multiplex PCR to evaluate the expression of sialyltransferase(ST) and fucosyltransferase (FucT) gene families in lung cancer (in >50% of patients), we have demonstrated that overexpression of STs and FucTs are common in lung cancer tissues and cell lines. Daughter cell line (CL1-5) with high invasiveness and metastasis ability expressed
more STs and FucTs than parent cell line (CL1-0 and CL1-1). Clinically, patients
bearing tumors with overexpression of ST3GalIII and FucTIII、IV、VI、VII are liable to cancer recurrence and death. We thus propose a three-year study to comprehensively study the role of glycosyltransferase genes expression in lung cancer. Is up-regulation or down-regulation of certain glycosyl -transferase which leads to expression of cancer-associated sialylated antigens occurs preferentially in lung cancer? Can the altered expression of glycosyltransferase genes serve as a prognostic marker? Specific glycosyltransferase genes considered to be important in lung cancers will be cloned and subjected to further study. In the first year, we evaluated the expression of N-acetyl -glucosaminyltransferase (MGAT) I~V in lung cancer and the association of MGAT expression with clinical features and prognosis of lung cancer.
Keywords: Lung cancer, glycosyltransferase
二、緣由與目的
Aber r ant glycosylation of mucins
Mucin glycoprotein consists of a protein backbone with many carbohydrate side chains of varying lengths, sequences, compositions and anomeric linkages. They have a very large molecular weight (400 to > 1000 kDa), many O-glycosidically linked carbohydrate side chains which may constitite 50-85% of the total molecular weight, a high content of serine, threonine and proline in the protein backbone structure. The carbohydrate moieties of mucin glycoprotein may provide important biological functions of cells. These include receptor function for growth factors, hormones, toxins, bacteria, and virus lectins, growth regulation, cellular differentiation, homotypic and heterotypic, cell-cell interaction, cell-substratum or cell-basement membrane interactions and various immunological functions.[1] Experimentally, mucins had been demonstrated to promote tumor cell invasion,
metastasis and modulate the immune recognition phenomenon of cancer cells.[2] The aberrant expression of mucins and mucin related antigens are poor survival factors in carcinomas arising from various organs, such as colon and breast cancer. Our previous studies had demonstrated the prognostic implication of mucins for lung cancer. [3-5] Lung cancers overexpressing sialomucins (highly sialylated mucins not so heavily expressed in normal airway) tend to have higher chance of recurrence and metastasis. The overexpression of sialomucin correlated with erbB-2/neu oncoprotein overexpression and at least one mucin core peptide (MUC5AC apomucin).
Aberrant glycosylation of mucins is common in cancer cells. Among cancer-associated antigens, sialylated Lewis antigens such as sialyl Lewis x (sLex) and sialyl Lewis a (sLea) have been well characterized. It is well known that the sLex and sLea epitopes produced in cancer cells are mainly carried on mucin O-glycans. The augmented expression of sLex and sLea antigens is frequently observed in some cancerous tissues, including lung cancer. It has been reported that sialyl Lex and sialyl Lea are involved in the process of metastasis, because these compounds serve as ligands for P- and L-selectin expressed on the surface of vascular endothelial cells, and mediate the adhesion of malignant cells to the vascular endothelium[6] Clinical reports also recognized these compounds to be poor prognostic factors for lung cancer and colon cancer.[7,8]
Glycosyltr ansfer ase and mucin glycosylation
Glycosyltransferases are enzymes arrayed in Golgi apparatus.. These enzymes transfer glycosyl residues from nucleotide-activated sugar molecules to other carbohydrates or aglycans (peptides or lipids) in a highly efficient and specific way. They work like sequential part of an assembly line with “cooperative sequential specificity”, that the product of one glycosyltransferase becomes the acceptor substrate for the next glycosyltransferase. By estimation, there are more than 250 glycosyltransferases, and the assembly of these enzymes on Golgi may be tissue specific, i.e. different tissue may have different glycosylation process on same peptide backbone.[9,10]
Mucin-type glycoproteins are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine in a polypeptide. The initiation of O-linked glycosylation occurs through the action of UDP-Gal-Nac: polypeptide N-acetylgalactosaminyltransferase (ppGaN-Tase), which catalyzes the transfer of GalNAc from the nucleotide sugar UDP-GalNAc to the hydroxyl group of either serine or threonine. [11]
These attached O-glycans can be classified into several different groups
according to the core structures subsequently added on GalNAc [12]. In many cells, core 1, Galβ1→3GalNAc, is the major constitute of O-glycans, core 1
oligosaccharides are converted to core 2 oligosaccharides, Galβ1→3 (GalNAcβ1 →6) GalNAc when core 2 β1,6-N-acetylglucos aminyltransferase (C2GnT) is present. [13] In the gastrointestinal tract and breast tissue, oligosaccharides with core 3,
GlcNAcβ1→3 GalNAc, can be frequently found. These tissues also contain core 4, Galβ1→4Glcβ1→6(GlcNAcβ1 →3)GalNAc. Core 4 is formed from core 3 by core 4 β1,6-N-acetyl glucosaminyltransferase (C4GnT). It has been reported that the amount of core 4 oligosaccharides is reduced in cancer cells, while the amount of core 2 oligosaccharides is maintained or increased.[14] More recently, the increase in the transcript of C2GnT was found to be associated with the progression of colonic carcinoma.[15]
A set of glycosyltransferases are required for the synthesis of cancer-associated sialylated antigens, they are: β1,3 N-acetylglucosaminyltransferase (β1,3GnT),
β1,4-galactosyltransferase (β1,4GalT), α2,3-sialyltransferase (ST3Gal), and
α1,3-fucosyltransferase (α1,3Fuc-T) for sLex synthesis, and β1,3GnT, β1,3 GalT, ST3Gal, and α1,4Fuc-T for sLea synthesis. The genes encoding β1,3GalT and
β1,3GnT, which are required for the synthesis of type-1 (Lea and Leb) and type-2 chains (Lex and Ley), have recently been cloned. The genes encoding β1,4 GalTs(I-VI), ST3GalTs (I-IV), α1,3Fuc-T (III-VI), α1,4FucT (FucTIII) and Core 1-4GnTs had been cloned.
Glycotr ansfer ase gene expression and cancer
Detailed mechanisms involved in regulation of glycosyltransferase genes are largely unclear. Most of the glcosyltransferase genes ("glyco-genes") are expressed in an organ- and tissue-specific manner [16]. For example, the 5' untranslated region of GlcNAc-TV mRNA from various cells showed multiple sequences depending on the cell types [17]. Analysis of the 5' untranslated region revealed the presence of consensus motifs for transcription factors such as TATA box, AP-1/Ets, AP-2, HNF-1, and HP-1. The gene employs a multiple promoter system for transcription, and gene expression may then be regulated in a tissue-specific fashion. More over, multiple GlcNAc-TV transcripts were reported in various cell lines and tissues [18]. Disruption of the GlcNAc-TV gene in mice results in the loss of any detectable GlcNAc-TV activity, as well as β1-6 GlcNAc branching glycans in all tissue examined [19]. GlcNAc-TV homozygous null mice are viable and fertile, but showed enhanced rates of T cell aggregation and cell proliferation. Enhanced
was also noted [20]. GlcNAc-TV catalyzes the attachment of a β1-6GlcNAc residue, producing branched N-glycans. The high degree of branching of N-glycans appears to be related to the malignant potential of tumor cells. In particular, β1-6 branching of N-glycans is directly linked to tumor metastasis. Transfection of mammary carcinoma cell lines with a GlcNAc-TV expression vector increased metastasis by 4-40 folds [21]. The expression of GlcNAc-TV gene could also be induced by oncogene (e.g., ras-signaling pathway) or viral (e.g., v-src) transfections during hepato-carcinogenesis. In addition, the GlcNAc-TV activity is also augmented by phorbol esters and TGF-β1. Recently, erbB-2/neu has been shown to be able to stimulate the transcription of GlcNAc-TV through Ras-Raf-Ets signal transduction pathway [22].
Unlike GlcNAc-TV, the molecular basis of most human glycogenes remains elusive. As the genomic regions containing the promoter were isolated in only few genes, and complete nucleotide sequence of many genes had only recently become available.
Rationale of study design
Our previous studies had demonstrated the prognostic implication of mucin expression for lung cancer. Lung cancers overexpressing sialomucins tend to have higher chance of recurrence and metastasis. The expression of sialomucin is correlated with the overexpression of an oncoprotein, erbB-2/neu, and at least one mucin gene (MUC5AC apomucin). We are than interested about the altered expression of glycosyltransferase genes in lung cancer. Preliminarily, using multiplex PCR to evaluate the expression of sialyltransferase(ST) and fucosyltransferase (FucT) gene families in lung cancer (in >50% of patients), we have demonstrated that overexpression of STs and FucTs are common in lung cancer tissues and cell lines. Daughter cell line (CL1-5) with high invasiveness and
metastasis ability expressed more STs and FucTs than parent cell line (CL1-0 and
CL1-1). Clinically, patients bearing tumors with overexpression of ST3GalIII and
FucTIII、IV、VI、VII are liable to cancer recurrence and death.
Expression of the above glyco-genes has also been studied in many other types of cancer, and also demonstrated tissue-specificity. For example, downregulation of FucT VII and ST3GalIII was noted in colon cancer and cervical cancer [23,24], while the opposite was shown in gastric cancer [25] and lung cancer (as in our preliminary data). Based on the PCR analyzing the CL cell lines, we noticed a successive increased expression of FucTIII, FucTVI and ST3Gal III from cell line with low invasiveness(CL1-0) to high invasiveness(CL1-5). Since the expression of
of glycosyltransferases is strongly correlated with mRNA expression, measured by either Northern blot or in situ hybridization [26]. We hypothesize that increase expression (activity) of FucT III, FucT VI and ST3Gal III play important role in lung carcinogenesis.
Both STs (ST3s and ST6s) and FucTs (FucT-III-VII) are key enzymes participating in synthesizing Lewis and sialyl-Lewis antigens (Lea, Leb, Lex, Ley, sLex, sLea). Lewis and sialyl-Lewis antigens are involved in the process of metastasis, these sialylated and fucosylated compounds serve as the ligands of E-selectin or P-selectin expressed on the surface of vascular endothelial cells. ST3GalIII preferentially synthesize sLea, FucTIII-VII are enzymes catalyzing the last step of synthesis of Lewis and sialyl-Lewis antigens. FucT III is the Lewis enzyme and ubiquitously expressed in the epithelial cells of aerodigestive tracts, and is the only FucT with both α1,3 and α1,4 fucosylation activity to synthesize both
α1,3-fucosyl-containing Lex and sLex and α1,4-fucosyl-containing sLea. FucT VI and FucT VII preferentially synthesize Lex, Ley, and sLex. FucT VI possesses the strongest activity in synthesizing sLex antigen.
ST3GalIII and FucT III are identified as genes of interest. Therefore, we are interested in studying whether the abnormal activation of ST3GalIII and FucT III may contribute to the metastatic phenotype during the progression of cancers. We will clone ST3GalIII and FucT III genes and establish inducible expression system of ST3GalIII and FucT III in transfected cell lines. We propose to transfect
ST3GalIII and FucT III sense cDNA into CL1-0 and anti-sense cDNA to CL1-5.
Biochemical or functional change of cells with overexpressed or down-translated enzymes will be evaluated by flow cytometry (formation of specific glycans, i.e. Lex ,sLex and sLea ), migration/ invasion/metastasis assay, anchorage-independent growth assay, in vivo tumorigenicity and metastasis. Catalytic activity of
glycosyltansferases in tranfected cell lines will also be measured.
Besides ST3s and FucTs, several classes of glycosyltansfease genes may have contribution to tumorigenesis. These genes include core1-4GnTs、β-1-4GalT I~VI、 GlcNAcTs (I-VI, especially III and V) [16,29]. Unlike STs and FucTs which modify terminal glycosylation, coreGnTs、β-1-4GalTs and GlcNAcTs synthesize the core glycan and elongate backbone glycans. Altered expression of these genes may affect cell surface receptors (e.g. cytokine and adhesion receptors) and influence cellular growth and proliferation. To identify candidate genes related to lung carcinogenesis, multiplex RTp-PCR of coreGnTs、β-1-4GalTs and GlcNAcTs will be performed in CL cell lines and in clinical lung cancer specimens.
Patients and tumor tissues
From 1992 to 1996, we have collected 309 pairs of lung tumor/nontumor lung tissues. Among them, 235 had the pathology of non-small cell lung cancer. Seventy tissue pairs from patients with detailed clinical data and follow-up history were chosen.
Surgical specimens of tumors and the adjacent uninvolved lung tissue will be obtained from patients with lung cancer at the time of resection. All patients should have non-small cell lung cancer confirmed by histological diagnosis. Preoperative staging work-up included chest radiograph, fiberoptic bronchoscopy with
brushing/washing cytology and biopsy, sputum cytology, computed tomography of thorax and abdomen, and bone scanning. Computed tomography of the brain is not a routine staging procedure. All patients have been judged preoperatively to have resectable disease, and all undergo a complete resection of the tumor. The resection will be judged complete if all known tumorous tissue is completely removed, resection margins are microscopically free of the tumor, and the area or nodes proximal to the involved area or nodes is microscopically free of tumor. After excision, tumor samples and the uninvolved lung tissues were collected
immediately, snap frozen in isopentane at –60°C placed in sterile jar and stored at –70°C until processed. Specimens used for formalin fixation and for OCT embedding are collected separately from specimens used for RNA isolation. The resected lung and lymph nodes were subjected to routine surgical pathological examination. Representative sections required for staging and histologic classification were generously taken. Sections of 4µm thickness were routinely stained with hematoxylin-eosin. Histologic classification was based on World Health Organization criteria. The final staging of each patient was pathologic, according to the international staging system for lung tumors.
Laser captur e micr odissection and RNA extr action
For microdissection, a laser microscope system (PixCell II™ LCM System, Arcturus Engineering, Inc., Mountain View, CA) is used. Areas containing tumor cells or cells from normal lung tissues for control are identified on unmounted H&E-stained section and are visualized through video monitor. CapSureTM film carriers are positioned over the cells of interest and capture the cells after a short pulse of low power infrared laser. The CapSureTM film carriers, with the attached cells, are lifted and placed into Eppendorf tubes and subjected for RNA extraction.
Multiplex PCR for glyco-genes
The expression of selected glyco-genes or gene family in tissues will be evaluated using multiplex PCR [31]. CDNA is synthesized from the isolated total RNA by
reverse transcription in 20 µl reactions containing 0.5µg of random primers, 200 U of superscript RTse, 2µg of total RNA, 4 µl of 5X RT buffer, 0.1mM each dNTP, 20 units Rnasin, and 6.5 µl DEPCed water. Each tube is incubated at room temperature for 10 min and then at 42°C for 45min, heated to 90°C for 10 mink, and then quick-chilled on ice. CDNA clones of each genes will be used as positive control. The PCR primers are designed based on the cDNA sequence of specific gene, and are selected with Primer 3 (a web-based primer designed, Whitehead Institute for Biomedical Research). In designing the primers for multiplex PCR, the possibility of primer dimerization is minimized by cross-examination of the mismatches in the sequences of each possible paur of all the primers chosen. Primers with optimal annealing temperatures of 56-60°C are chosen to prevent mismatches, and primers with higher temperatures are chosen for shorter PCR products. Each PCR is performed in a 100-µl reaction mixtuer containing 10 µl RT reaction mixture, 1X PCR buffer, 0.1mM of each dNTP, 2.5 units of Taq polymerase, adjusted
concentration of each primers, and subjected to 28-30 cycles of PCR reaction. The PCR reaction products are then electrophoresed in a 3% agarose gel and stained with ethidium bromide, and the size of each cDNA product is determined by comparison to size marker (100bp DNA ladder).
Quantitaiton of multiplex RT-PCR products
After amplification, the PCR products were electrophoresed in 3% agarose gel, stained in 5 µg/ml ethidium bromide solution for 30 min, and then destained. The signal intensity of amplified native and mutated products was directly measured and digitized by IS-1000 digital imaging system (Alpha Innotech Incorp., San Leandro, CA). To evaluate the relative levels of expression of the target genes in multiplex RT-PCRs, the value of the internal standards (β2-microglobulin, GAPDH, or β-actin) in each test tube is used as the baseline gene expression of that sample, and the relative value is calculated for each of the target genes amplified in that reaction. These values are then used to compare expression across samples tested.
四、結果
The expression of N-acetylglucosamin- ylransferase (MGAT, or GnT) I~V was evaluated in 49 paired tissues of lung cancer patients. The enzymes have broad acceptor activity for both N- and “mucin-type” O-glycan branching activity. There were 30 adenocarcinoma, and 19 squamous cell carcinoma. Stage distribution was: 20 stage I, 10 stage II, 17 stage III; 9 T1, 36 T2, 2 T3, 2 T4; 24 N0, 10N1,14 N2, 1 N3.
Overexpression of MGAT(GnT)s in lung cancer is not rare: MGAT I (57.1%), MGAT II (69.4%), MGATIV (40.8%), MGAT V (36.7%).
10
The correlation between MGATs expression and the clinical features or outcome of lung cancer patients reveals that only the expression MGAT1 has correlation with histology (78.9% of squamous cell carcinoma overexpressed MGAT1, vs 43.3% of adenocarcinoma, P=0.014). Patients bearing tumors with MGAT1 overexpression tended to have earlier disease relapse and cancer death. (Fig 1A and 1B). Survival Functions RELAPSE 3000 2000 1000 0 Cu m S ur vi va l 1.0 .8 .6 .4 .2 0.0 Mgat1 T/N raio >1 ratio>1 ratio>1-censored ratio<1 ratio<1-censored
Fig 1A. Kaplan-Meier curve of tumor relapse according to the status of MGAT1 expression.
Survival Functions S ur vi va l 1.0 .9 .8 .7 .6 .5 .4 .3 .2 Mgat1 T/N raio >1 ratio>1 ratio>1-censored
五、參考文獻
1. Gendler SJ and Spicer AP. Epithelial mucin genes. Ann Rev Physiol. 1995;57:607-34.
2. Van Klinken BJW, Dekker J, Buller HA, and Enerhand AWC. Mucin gene structure and expression: protection vs. adhesion. Am J Physiol.
1995;269:G613-27.
3. Yu CJ, Yang PC, Shew JY, Hong JM, Yang SC, Lee YC, Lee LN, Luh KT, Wu CA. Mucin mRNA expression in lung adenocarcinoma cell lines and
tissues.Oncology 1996;53:118-26.
4. Yu CJ, Yang PC, Shun CT, Lee YC, Kuo SH, Luh KT. Overexpression of MUC5 genes is associated with early postoperative metastasis in non-small cell lung cancer. Int J Cancer 1996; 69:457-65,
associated with erbB-2 oncoprotein overexpression, early recurrence and cancer death in non-small cell lung cancer. Am J Respir Crit Care Med.
1997;155:1419-27.
6. Shimizu Y and Shaw S. Mucins in the mainstream. Nature 1993;366:630-1.
7. Ogawa Ji, Sano A, Koide S, and Shohtsu A. Relation between recurrence and expression of proliferating cell nuclear antigen, sialyl LewisX, and sialyl Lewis(a) in lung cancer. J Thorac Cardiovas Surg 1994;108:329-36.
8. Ogawa Ji, Sano A, Inoue H, and Koide S. Expression of Lewis-related antigen and prognosis in stage I non-small cell lung cancer. Ann Thorac Surg
1995;59:412-5.
9. Van den Steen P, Rudd PM, Dwek RA, and Opdenakker G. Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol Biol 1998;33: 151-208.
10. Kleene R and Berger EG. The molecular and cell biology of glycosyltransferases. Biochem Biophys Acta 1993;1154:283-325.
11. Ten Hagen KG, Hagen FK, Balys MM, Beres TM, Van Wuyckhuyse B, and Tabak LA. Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family. J Biol Chem 1998;273:27749-54.
12. Yeh JC, Ong E, and Fukuda M. Molecular cloning and expression of a novel beta-1, 6-N-acetylglucosaminyltransferase that forms core 2, core 4, and I branches. J Biol Chem 1999;274: 3215-21.
13. Fukuda M, Carlsson SR, Klock JC, and Dell A. Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells. J Biol Chem 1986;261:12796-806.
14. Yang JM, Byrd JC, Siddik BB, Chung YS, Okuno M, Sowa M, Kim YS. Mtta KL, and Brockhausen I. Alterations of O-glycan biosynthesis in human colon cancer tissues. Glycobiology 1994;4:873-84.
15. Shimodaira K, Nakayama J, Nakayama N, Hasebe O, Katsuyama T, and Fukuda M. Carcinoma -associated expression of core 2
beta-1,6-N-acetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res 1997;5201-6.
16. Taniguchi N, Jain SK, Takahashi M, et al. Glycosyltransferases: cell surface remodeling and regulation of receptor tyrosine kinase-induced signaling. Pure Appl. Chem. 1999;71:719-28.
17. Saito H, Gu J, Nishikawa A, Ihara Y, Fujii J, Kohgo Y and Taniguchi N. Organization of the human N-acetylglucosaminyltransferase V gene. Eur J
Biochem 1995;233:18-26.
18. Perng GS, Shoreibah M, Margitich I, Pierce M and Fregien N. Expression of N-acetyl-glucosaminyltransferase V mRNA in mammalian tissues and cell lines. Glycobiology 1994;4: 867-71.
19. Granovsky M. Fode C. Warren CE. Campbell RM. Marth JD. Pierce M. Fregien N. Dennis JW. GlcNAc-transferase V and core 2 GlcNAc-transferase expression in the developing mouse embryo. Glycobiology. 5:797-806, 1995
20. Dennis JW. Granovsky M. Warren CE. Protein glycosylation in development and disease. BioEssays. 1999;21:412-21.
21. Seberger PJ. Chaney WG. Control of metastasis by Asn-linked, beta1-6 branched oligosaccharides in mouse mammary cancer cells. Glycobiology 1999;9:235-41.
22. Chen L, Zhang W, Freigen N, and Pierce M. The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products. Oncogene 1998; 17:2087-93.
23. Geβner P, Riedl S, Quentmaier A, Kemmner W. Enhanced activity of
CMP-NeuAc:Galβ1-4 GlcNAc:α2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett 1993;75:143-9.
24. Wang PH, Li YF, Juang CM, et al. Altered mRNA expression of sialyltransferase in squamous cell carcinoma of the cervix. Gynecol Oncol 2001;83:121-7.
25. Petretti T, Schulz B, Schlag PM, Kemmner W. Altered mRNA expression of glycosyltransferases in human gastric carcinomas. Biochem Biophys Acta 1999;1428:209-18.
26. Harduin-Lepers A, Krzewinski-Recchi MA, Hebbar M, et al. Sialyltransferases and breast cancer. Recent Res Dev Cancer 2001;3:111-26.
27. Liu F, Qi HL, Chen HL. Regulation of differentiation-and proliferation-inducers of Lewis antigens, a-fucosyltransferase and metastatic potential in
hepatocarcinoma cells. Br J Cancer 2001;84:1556-63.
28. Seidenfaden R, Hildebrandt H. Retinoic acid-induced changes in
polysialyltransferase mRNA expression and NCAM polysialylation in human neuroblastoma cells. J Neurobiol 2001;46:11-28.
29. Dennis JW, Granovsky M, Warren CE. Protein glycosylation in development and disease. BioEssays 1999;21:412-21.
30. Chu YW, Yang PC, Yang SC, et al. Selection of invasive and metastatic subpopulation from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997;17:353-60.
31. Wei Q, Xu X, Cheng L, Legerski RJ, Ali-Osman F. Simultaneous amplification of four DNA repair genes and β-actin in human lymphocytes by multiplex reverse transcriptase-PCR.Cancer Res 1995;55:5025-9.
32. Yu D, Wang SS, Dulki KM, Tsai CM, Nicolson GL, Hung MC. C-erbB2/neu overexpression enhances metastatic ppotential of human lung cancer cells by induction of metastasis-associated properties. Cancer Res 1994;54:3260-6.
