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Life Science Journal, 3 ( 3 ) ,2006 , Ba , et aI, Mage-a x , rnRNA Level in Lung Cancer by Coal Tar Pitch

Mage-ax mRNA Level in Lung Cancer of Mice Derived

by Coal Tar Pitch

Yue Ba, Huizhen Zhang, Qingtang Fan, Xiaoshan Zhou, Yiming Wu

Department of Labor and Environmental Health, College of Public Health,

Zhengzhou University, Henan 450052, China

Abstract: Objective. To investigate the expression of Mage-ax mRNA in lung cancer tissues of mice induced by coaltar pitch (CTP) fume and to discuss the pos",ibility of the lung cancer animal model induced by CTP as a model forlung cancer immunotherapy with MAGE-A. Methods. Tumor tissue samples of lung cancer and paired non-tumortissues of the lung were obtained from 8 lung cancer mice. Total RNA was extracted and cDNA was synthesized.Nested PCR amplification using Mage-ax specific primers was then performed to detect the expression of Mage-a.

The 2 clones of 1 sample of Mage-ax mRNA positive PCR products were DNA sequenced by using DNA" sequencer(PE-377). Results. Of 29 mice in the experimental group, 8 were induced to lung cancer. Of 8 lung cancers, 5(62.5%) expressed Mage-ax mRNA. The expression of Mage-ax gene was not recognized in adjacent lung tissuesat all. The DNA sequence confirmed that the target gene fragments in all 2 samples of PCR products were Mage-ax

cDNA. Conclusion. The Mage-a gene was expressed highly in tumor tissues with lung cancer in mice induced byCTP fume. This suggests that this kind of lung cancer mouse model may be an ideal animal model for lung cancertherapeutic experiment by MAGE-A. [Life Science Journal. 2006; 3(3) : 29 - 34] (ISSN: 1097 - 8135) .

Keywords: Mage-ax mRNA; mouse; CTP fume; lung cancer

Abbreviatious: CTP: coal tar pitch; CTL: cytotoxic T lymphocyte; TAs:tumor antigens

1 Introduction

Although the enormous manpower and materi-al resources have been spent, there are still no ef-fective methods developed to prevent and treat ma-lignant tumor. The incidence rate of malignant tu-mor is increasing and the onset age is tending to beyounger along with the changes of environment andlifestyle of human being. In China, the increasingmagnitude of lung cancer is in the first place in re-cent 20 years according to the infonnation derivedfrom a more recent national conference on oncologyin 2000[1].

Using a gene transfection approach to identifyantigens recognized by CTL (cytolytic T lympho-cytes) on a human melanoma cell line, Boon et al

isolated the gene MACE-A family that is located inthe Xq28 region and the gene family includes at

least 12 related genes[2-4J. MACE-B, including 4

genes, was identified in the Xp21. 3 region[5-7J.MACE-Cl is on band Xq26[8J. Most of theseMACE genes are expressed in a significant propor-tion of tumors of various histological origins,whereas no expression has been observed in normaltissues except on placenta and male genn cells.

The MACE-encoded antigens are recognizedby cytolytic T cells in the fonn of antigenic pep tides

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presented by HLA class I molecules. Because malegenn cells do not express the HLA class I genes,they fail to present MAGE antigens even thoughthey express MACE genes. The MACE-encodedantigens are therefore strictly tumor-specific. Sev-eral immunogenic peptide epitopes from tumor-as-sociated antigens (such as MAGE-A3, MAGA-A1etc. ) have served as targets for cellular immune re-sponses in numerous clinical trials for therapeuticvaccinations[9, 1OJ.

In 1999, Boon et al found Mage-a, a newI ilmily of mouse genes homologous to the human

MACE-A genes[llJ. Mage-a genes were mappedon X chromosome. Like human MACE-A, Mage-a genes were transcribed in adult testis, but not inother tissues. Expression of some Mage-a geneswas also detected in tumor cell lines. Mage-a genesare higher degree homologous to the humanMACE-A genes. Like MACE-A genes, theyen-code acidic proteins. As the ideal tumor animalmodel, it is possible to research the immunotherapyby using MACE tumor antigens(TAs). There is,however, little information on their expression inlung carcinoma of mice. This experiment studiedthe expression of Mage-a gene in mice lung cancerand compared its sequence with that in GenBank,then discussed the possibility of the lung cancer ani-

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nal elongation cycle at 72 °C for 300 sec. Under thesame PCR condition, a second round of PCR was

performed using nested primer 5' -AGCGGATCC-CTCTCTCCCCAGGCC-3' (forward), 5' -A C G

bAGCTTCCAATTTCCGACGACACTCC-3' (re-verse) , and 1 t-tl of first-round PCR products astemplate. The nested primers were added appropri-ate BamHI and Hind III restricted site respective-

ly.2.3mids

For the construction of recombinant pUC18

plasmids, a PCR fragment of Mage-ax cDNA wasobtained with the nested primer. pUC18 plasmidsand the target fragments were digested by BamHIand Hind III respectively. Bands were isolated fromlow melting point agarose and purified by UNIQ-5column DNA gel reextraction kit (Sangon, Shang-hai, China) respectively, and then PCR productwas ligated directly into pUC18 plasmid with T4

DNA ligase. The mixture was transfered into E.coli cell, strain DH5a according to the method of

Molecular Cloning[12]. At least 3 positive cloneswere picked up and amplified. Then, the ligatedPCR products were isolated and sequenced bydideoxy method using DNAs sequencer (PE377).The sequencing results were analyzed using DNAsissoftware.

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Life Science Journal, 3 (3) ,2006, Ba , et al, Mage-ax, mRNA Level in Lung Cancer by Coal Tar Pitch

mal model induced by CTP as a model for lung can-cer immunotherapy with the use of MAGE-A.

Mage-al' a2, a3, as, a6, as of Mage-a are ar-ranged in a cluster located in a region syntenic toXp22 and they share more than 93 % nucleotide identi-ty. The above 6 genes which were amplified in thisstudy were called as Mage-ax'

2 Materials and Methods

2.1 Sample collection and RNA extractionAnimal: 64 Kunming mice, 32 males and 32

females, were provided by Henan Animal Center(Zhengzhou, Henan, China). The mice were di-vided into experiment group and control group ran-domly. The experiment group was exposed to CTPfume 2 h per day for 12 weeks. The mice werekilled in the 12th week and the 24th week, respec-tively. The lung tissues were frozen in liquid nitro-gen. All the samples were confirmed by pathology.

Total cellular RNA was isolated using the flashcolumn total RNA preparation kit (QIAGEN, Ger-man) according to the manufacture's instructions.2.2 Nested RT-PCR

For nested RT-PCR analysis of Mage-ax tran-scripts, 5 t-tgof total RNA were reverse-transcribedwith the first round PCR specific primers: 5'-AATACCAAGTCCTCCCCAG-3' (forward), 5'-CTTGGGCCCCACAGGAACC-3' (reverse) in a30 t-tlreaction mixture containing reverse transcrip-tase buffer, 5 mmol dNTP, 25 pmol primer and10 U AMV reverse transcriptase (Promega, USA).The mixture was incubated at 42°C for 60 min,heated at 95°C for 5 min and then stored under -

20°C. 3 t-tlof RT reaction were used in one roundof PCR with 1 X Taq buffer, 5 mmol dNTPs, 25pmol first round PCR specific primers and 2 U ofTaq DNA polymerase (Promega, USA). PCR am-plification was 35 cycles at 94°C for 50 sec, 55°Cfor 50 sec and 72°C for 60 sec with an initial one-cycle predenaturation at 94°C for 120 sec and a fi-

Cloning and sequencing of recombinant plas-

3 Results

3. 1 The results of CTP induced cancerOf 29 mice in the experiment group, 8 were

induced to lung cancer, 7 were carcinoid and'l wasadenocarcinoma within 24 weeks. There were 5specimens with squamous metaplasia, 2 were hy-perplasia and the rest were normal. All the 32 micein control group were normal. The cancer incidencewas significantly different between two groups (P< 0.01). Detail results were shown in Table 1.

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Experiment groupP<O.Ol

C'AJmparisonof tumor induced in control group and experiment group

Cases( n) Mice with tumor( n) Mice without tumor( n)

TI 0 TI

29 8 21

Table 1.

3.2 Expression of Mage-ax gene at mRNA levelin mice lung cancer tissues

Of 8 mice with cancers, Mage-aT mRNA wasexpressed in 5 (62. 5% ). The expression of Mage-ax gene was not recognized in adjacent and normallung tissues at all. Mage-ax gene was also ex-

Ratio

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pressed' in one squamous metaplasia. The PCRproducts were then digested by restriction endonu-cleas Sca I. The two fragments, 300 bp and 200bp, were shown respectively. Representative gelswere shown in Figure 1 and Figure 2. Results wereshown in Table 2.

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- 500 bp

Figure 1. Amplification of target gene

Amplified product of Mage-ax cDNA was 492 hp.

Lane 1,2,3,4: lung cancer tissues which expressed

MaKe-aT mRNA: Lane M: marker

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Sea I ; Lane M: marker

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Table2. Expression of MACE-ax gene in lung tumors inmice

Pathology

Carcinoid

Adenocarcinoma

Squamous metaplasia

Hyperplasia

Adjacent lung tissues

Nonnallung tissues

Proportion of positive samples

4/7 (57.1%)

1/1 (100%)

1/5 (20%)

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3.3 Construction, identification and sequencingof mouse Mage-ax gene

The amplified DNA fragment was digestedwith BamHI and Hind ill. The target fragmentwas ligated into a predigested (with BamHI andHind ill) clone vector pUC18. Initial transforma-tion was carried out with E. coli DH5a hoststrain. Positive clone was identified via preparing

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the plasmid DNA from a number of clones and ana-lyzed by using amplification with universal primerof pUC18 on agarose gel electrophoresis, and wassequenced by using primer M13 (or pUC18 univer-sal primer). The target gene fragments in samplesof PCR products were Mage-ax cDNA. The resultswere shown in Figure 3 and Figure 4.

4 Discussion

The identification of TAs and their recognitionby tumor-specific CTL has fuelled the developmentof immunotherapeutic strategies in cancer[13]. Al-though numerous TAs and their epitopes have beenidentified, the majority of these are quite restrictedin expression and their clinical utility remains limit-ed. Therefore, it is imperative to evaluate the pos-sibilityof tumor immunotherapy by using TAs/epi-topes that are widely expressed in tumor. It hasmany benefit to research the MACE genes' func-tion in mice such as sample got easier, dynamic ob-servation and so on. It may, therefore, be the idealanimal model for the lung cancer's therapeutic ex-periment by using MACE.4. 1 Reliability, sensitivity and specificity of theexperiment

The authors took the following measures toensure the reliability of the experiment: (1) Re-move the necrosis tissues to refrain from RNAdegradation caused by necrosis. (2) RNA extractedwas all verified by ethidium bromide fluorescenceelectrophoresis and by ultraviolet radiomete (A260/A280>1. 9). (3) To ensure RNA was not degrad-ed, a PCR assay with primers specific for ~-actinwas carried out in each case. (4) To avoid the falsepositive results, all the samples of extracted RNAwere incubated with DNase. (5) Random samplingthe positive clones sequenced and confirmed theamplified products being Mage-ax (The sequence isthe same as that of GenBank).

Method of nested PCR can improve the sensi-tivity and specificity of the experiment. The firstround PCR will be carried out using the exo-primers firstly, and then the second round PCRwill amplify the smaller regions of the first roundPCR products by using the nested-primers. So thecontinuous twice enlargement may increase both thesensitivity and the specificity of PCR greatly. Forthe extreme-trace target fragments, it's very diffi-cult to get the better results for once amplification,but satisfactory results can be obtained using nestedPCR.

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Figure 3. Identification of recombinant-plasmid amplified by pUC18 universal primerLane 1,2,3,4,5,7: the inserted target fragments amplified by universal primer; Lane 6: plasmid without inserted the target gene;

Lane 8 : the negative control; Lane 9: plasmid inserted the target gene

Smageal :Smagea2 :Smagea3 :SmageaS :Smagea6 :Smagea8 :5MBY1

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4.2 Expression of Mage-ax mRNA of mouse lungcancer tissues

As the results of CfP fume inducing lung can-cer among 29 mice in the experiment group within24 weeks", 8 were induced to lung cancer, 7 werecarcinoid and 1 was adenocarcinoma, respectively.

PCR results showed that the majority of lung tu-mors in mice (5 of 8 tested) were positive for theMage-ax gene by gene-specific primers and thenested-PCR method. These results suggested thatmore than 60 % of lung cancers were expressedMage-ax gene, which was also expressed in one

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squamous metaplasia, but not recognized in adja-cent and nonnallung tissues at all. Although thereare some mouse models used to evaluate human im-

mune responses to .MAGE-based tumor vaccine,there is no other infonnation about the expressionof Mage-a in lung carcinoma in mice. Gravekampet at. found high expression levels of Mage-b (an-other Mage family of mouse genes homologous tohuman .MAGE-B genes) in almost all metastases,regardless of age. The expression levels were 2- to3- fold higher in the metastases than in the primary

4TO7cg breast tumors[14]. It suggests that Mage-encoded tumor antigen will be used as model tostudy various anti-tumor immunization modalities. . .

In VlVO.

.MAGE-A Ags were detected in primary andmetastatic tumors of various histological types in-cluding melanoma, lung, bladder, ovarian, andbreast carcinomas. Individual .MAGE-A expressionvaries from one tumor type to another, but overallthe majority of tumors express at least one of the.MAGE-A family. Targeting epitopes shared by all.MAGE-A Ags would be of interest against a broadspectrum of cancers. At present, .MAGE peptide-based vaccines have been used in clinical trials with

tumorous patients, but with limited success[15,16].

A suitabl~ animal tumor model that would pennitthe optimization of .MAGE -encoding cancer vac-cines in mice is very necessary to immunotherapy oftumors using .MAGE gene products. Researchershave used different tumor animal models to evaluate

the possibility of tumor immunotherapy by using.MAGE Ags. Eggert et at demonstrated the im-

munogenicity of two Kb-restricted peptide epitopesderived from mouse MAGE proteins which mayserve as valuable tool for preclinical evaluation of

vaccination strategies[17]. Ni found that rSFV vac-cine could elicit human MAGE-3-specific antibodyand CTL response in the Trimera mice[18]. The re-sults of Gravekamp indicated that the metastaticand nonmetastatic breast tumor models could be

useful model systems to analyze how breast cancervaccines for humans[14].

However, a suitable mouse tumor model of

lung cancer that would pennit the optimization of.MAGE-encoding cancer vaccines in mice is cur-rently not available. In this study, it shows thatMage-ax gene was expressed highly in tumor tis-sues with lung cancer induced by CTP fume. Thissuggests that this lung tumor mice expressingMage-ax may be an ideal animal model for lungcancer therapeutic experiment by using .MAGE-A.

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Correspondenceto:Yiming WuDepartment of Labor and Environmental HealthCollege of Public HealthZhengzhou UniversityZhengzhou, Henan 450051, ChinaTelephone : 86-371-6778-1797Email: ymwu@zzu.edu.cn

AcknowledgmentsThis work was finished in Key Molecular Lab

of Henan Province. We are particularly grateful toProfessor Yi Zhang (Ludwig Institute for CancerResearch, Brussels Branch, Belgium) for his greathelp.

This work is supported by grants from theKey Project of Scientific Committee of HenanProvince (No. 524410067) and the Natural Sci-ence Research Fundation of Henan Educational Bu-reau(No. 2006330003).

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peripheral T cell population to a cytotoxic T lymphocyte

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