Characterization of 67 kD laminin receptor, a protein whose gene is overexpressed on treatment of cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide.

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Characterization of 67 kD Laminin Receptor, a Protein Whose

Gene Is Overexpressed on Treatment of Cells with

Anti-Benzo[a]pyrene-7,8-Diol-9,10-Epoxide

She-Juan An,*

,

‡ Jia-Kun Chen,†

,1

Hua-jie Chen,* Wei Chang,* Yi-Guo Jiang,† Qing-Yi Wei,* and Xue-Min Chen*

,1

*Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430030, China; †Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou 510182, China;

and ‡Lung Cancer Institute, Research Center of Medical Sciences, Guangdong Provincial People’s Hospital Guangzhou 510080, China Received September 1, 2005; accepted January 6, 2006

The molecular mechanisms potentially related to tumorigenesis

induced by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE)

were investigated by suppression subtractive hybridization of the

human bronchial epithelial cells (16HBE) carcinoma induced by

BPDE-transformed 16HBE cells (16HBE-C). The 67 kD laminin

receptor gene (67LR1) is one of the screened overexpressed genes

in 16HBE-C cells when compared with 16HBE. In order to

un-derstand the main functions of 67LR1 gene, we amplified the full

length of 67LR1 gene using reverse transcription-polymerase

chain reaction (RT-PCR) method. The amplified gene products

were inserted into pcDNA

TM

3.1 Directional TOPO expression

vector. We then transfected 16HBE cells with this vector and

derived stable transfected 16HBE cell lines containing the 67LR1

gene by using lipofectin and G418 selection protocols. The

ex-pression products of transfected genes were analyzed by

semi-quantitative RT-PCR. Soft agar growth assay was carried out to

identify the malignant features of 67LR1 gene. The stable

trans-fected cell lines can form colonies in soft agar. Further, the

transfected cells showed morphological changes compared to the

control cells, such as the obvious pseudopods. These data suggest

that the 67LR1 gene may be related to malignant transformation

induced by the anti-BPDE. The 67LR1 protein may be related to

the directionality of cell movement.

Key Words: anti-BPDE; laminin receptor; gene; carcinogenesis;

pseudopod.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous

environmental contaminants. They are found in the air, soil,

water, and plants and also in food (Cavret et al., 2005;

Kulhanek et al., 2005; Skrbic et al., 2005). PAHs represent

a class of toxicological compounds, which can create a variety

of hazardous effects in vivo, including cytotoxicity,

genotox-icity, immunotoxgenotox-icity, teratogengenotox-icity, carcinogenesis, and

neu-rotoxicity (Abbott, 1995; Kerkvliet, 1995; Manz et al., 1991;

Puga et al., 2002; Tu et al., 2004). Recently, especially in

Western countries, there is increasing research interest in the

health effects of PAHs, because they may enter the food chain

through different sources such as the pollution of air,

un-derground water, and soil (Cavret and Feidt, 2005; Houessou

et al., 2005; Krishnamurthi et al., 2003; Lemiere et al., 2005).

Benzo[a]pyrene (B[a]P) is a representative member of PAH

family, and it undergoes metabolic activation after entering the

mammalian cells to highly toxic reactive metabolite

intermedi-ates, which can irreversibly damage cellular macromolecules

(i.e., DNA, proteins, and lipids) (Chen et al., 2000; Pavanello

et al., 1999; Rubin, 2001).

Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) is one of the many metabolites of B[a]P,

and the formation of anti-BPDE–DNA adducts is considered to

be critical in the carcinogenic process of B[a]P (Chen et al.,

2000; Pavanello et al., 1999). Although the ultimate carcinogen

anti-BPDE reacts with nuclear DNA and becomes mutagenic

(Rubin, 2001), the mechanisms for anti-BPDE-induced

carci-nogenesis are not fully understood. For a better understanding

of cellular behaviors, the identification of genes differentially

expressed in a certain type of cells exposed to carcinogens has

been of a great research interest. This study is carried out to

investigate the differentially expressed genes and their main

characteristics induced by anti-BPDE.

In our prior papers, we successfully established the

malig-nant transformation model of human bronchial epithelial

(16HBE) cells induced by anti-BPDE (Jiang et al., 2001).

The 16HBE cell line was a gift kindly from Prof. Jun Xu

(Guangzhou Institute of Respiratory Disease, Guangzhou,

China). The 16HBE cells were treated once or several times

by anti-BPDE at different doses, and the foci were observed and

assessed at the different stages during the period of the whole

experiment. The features of malignancy of the transformed

16HBE cells were identified by the test of soft agar culture.

The results showed that the best method for malignancy

trans-formation of 16HBE cells induced by anti-BPDE was that the

16HBE cells were treated several times with anti-BPDE at

1_{To whom correspondence should be addressed at Institute for Chemical}

Carcinogenesis, Guangzhou Medical College, Guangzhou, 510182, China. E-mail: cxm3636@yahoo.com.cn.

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concentration of 0.1, 0.5, 1.0, and 2.0 lmol/l and were passaged

fifteen times. The transformed cells could grow in soft agar and

grow into tumors in BALA/C nude mice. The tumors were

squamous cell carcinoma confirmed by histopathological

examination. We isolated the cells (16HBE-C) from the

carcinoma induced by anti-BPDE-transformed cells treated

four times at a dose of 2.0 lmol/l. Eight known genes were

differentially overexpressed in 16HBE-C cells compared to

16HBE cells using suppression subtractive hybridization (SSH)

method (An et al., 2005). Laminin receptor 1(67-kDa) (67LR1)

gene was one of the screened overexpressed genes in 16HBE-C

cells compared to 16HBE cells. The aim of the present study

was to investigate the main characterization of 67LR1 gene.

MATERIALS AND METHODS

Cell culture, isolation of total RNA, and mRNA suppression subtractive hybridization. The human bronchial epithelial cells (16HBE cells), a carci-noma cell line derived from transformed 16HBE cells (16HBE-C cells) induced by treatment with the anti-BPDE and suppression subtractive hybridization (SSH), were established in our laboratory as previously reported (An et al., 2005; Jiang et al., 2001). Briefly, The16HBE cells were treated four times with anti-BPDE at a concentration of 2.0 lmol/l and passaged 15 times. Then the malignant features of the cells were assessed by soft agar culture and tumorigenesis in nude mice. The 16HBE-C cells come from the carcinoma derived from transformed 16HBE cells induced by anti-BPDE. Both of the cell lines were cultured in 25-cm2 flasks with 5% carbon dioxide and 100% humidity at 37°C. The cells were grown in the MEM medium supplemented with 10% calf serum. Trizol (Gibco BRL, Carlsbad, CA) was used to extract the total RNA from cells, and the Oligotex Direct mRNA kit (QIAGEN Company, Hilden, Germany) was used to isolate the poly (A)þmRNA. The integrity of total RNA and mRNA were examined by electrophoreses of the samples on a 1% agarose gel, and the quantification and purity were analyzed by A260 and A260/280 (Eppendorf BioPhotometer, Germany), respectively. Suppression subtractive hybridization (SSH) was performed with 16HBE-C and 16HBE cells using the PCR-SelectTM_{cDNA Subtraction kit (Clontech, Palo Alto, CA)}

according to the manufacturer’s recommendations. The cDNA of 16HBE cells was used as the driver and 16HBE-C cells as the tester. After two times of subtractive hybridization and two times of PCR, the products of second nested PCR were inserted into TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and transferred into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). All of the positive clones were selected, sequenced, and compared with all sequences available in the National Center for Biotechnology Information (NCBI) nucleic acid database or EST database.

Full-length amplification of 67LR1 gene. Based on the screened nucleotide sequence from the suppression subtractive hybridization, the full-length sequence of Laminin receptor 1(67 kDa) (67LR1) gene was downloaded from GenBank. In order to match the overhang (GTGG) in the cloning vector pcDNATM

3.1 Directional TOPO, the nucleotide sequence 5#-CACC-3# was added to the forward primer, and the stop codon was removed. The gene-specific sequences of the primers are as follows: the forward primer, 5#-CACCATGTCCGGAGCCCTTGATG-3#; the reverse primer, 5#-TGCAA-GAACAGCTTAAGAC-3#. The product length of the amplification is 904 bp. ThermalAceTMDNA polymerase reaction was used to obtain the full-length gene. In order to get more purified products, gel purification of the PCR products was carried out according to the instructions of the manual (Nucleo-Trap Gel Extraction Kit, Clontech, Palo Alto, CA).

Construction of the expression vector and transfection of 16HBE cells. The products of the gel purification of the gene were inserted into

expression vector pcDNATM3.1 Directional TOPO (Invitrogen, Carlsbad, CA) and transformed into One Shot TOP10 chemically Competent Cells. Five colonies were randomly picked up and verified by PCR and DNA sequence analysis. Plasmid DNA of the positive colonies prepared using the QIAGEN Plasmid Purification Kit (QIAGEN Company, Hilden, Germany) was used to transfect 16HBE cells by the lipofectin transfection procedure. Stable trans-fected cell lines were selected using G418 (200 lg/ml). Overexpression of 67LR1 gene was determined by semiquantitative RT-PCR. The sequence of the gene specific primers used were 5#-AAACCCTGCTGATGTCAG-3#and 5#-ACAGATCAGGCATGACCTC-3#, These results were normalized using b-actin as the internal control, the gene specific primers of b-actin were 5#-GTGGGGCGCCCCAGGCACCA-3# and 5#-CTCCTTAATGTCACGCAC-GATTTC-3#. The length of 67LR1 and b-actin were 398 bp and 539 bp, respectively. The conditions of PCR were 20 cycles of denaturation at 94°C for 30 s, annealing at 49°C for 30 s, and extension at 72°C for 1 min.

Soft agar growth assay of the transformed cells. The ability of cell lines to grow in soft agar was measured by using the 16HBE cell line transfected with the 67LR1 gene. The 16HBE and 16HBE cell lines with transfected vector were used as negative controls. The 16HBE cell line with the transfected gene encoding the eukaryotic translation elongation factor 1a1 (eTEF1a1) was used as a positive control. eTEF1a1 gene is another differentially expressed genes in FIG. 1. The full-length amplification of 67LR1 gene from the RNA of 16HBE-C cells. M: DNA marker; 1 and 2: the results of 67LR1 gene (904 bp) amplified from the RNA of 16HBE-C cells.

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16HBE-C cells. Cells were seeded in 60-mm diameter dishes (1000 cells/dish) in 0.35% agar in complete medium on a base of 0.6% agar. There were five parallel culture dishes in every group. Colonies were counted 14 days later by microscope and by eyes, respectively.

RESULTS

Identification of Total RNA and Poly (A)

þ

mRNA Quality

and the Results of SSH

Total RNA exhibited two bright bands, which correspond to

28S and 18S RNA, respectively, with a ratio of intensities of

about 1.5–2.5:1. Poly (A)

þ

mRNA appears as a smear with

weak ribosomal RNA bands (An et al., 2005). The ratio of

A260/280 of total RNA and mRNA was greater than 2.1. The

results of SSH revealed that 67LR1 gene is one of the

overexpressed genes in 16HBE-C cells compared with

16HBE (An et al., 2005).

The Full-Length Amplification of 67LR1 Gene, Construction

of the Expression Vector, and Transfection of 16HBE Cells

The full-length 67LR1 gene (904 bp) was successfully

amplified from the RNA of 16HBE-C cells (Fig. 1), and the

amplified gene was inserted into the pcDNA

TM

3.1 Direc-tional TOPO vector. The positive transformants were

ana-lyzed by PCR, using the T7 and BGH priming site in the vector,

and were further confirmed by the sequencing analysis of

the transcription initiation region in the gene (Fig. 2).

Semi-quantitative RT-PCR analysis of gene expression showed that

the gene is overexpressed in 67LR1-transfected 16HBE cells

compared with the controls (Fig. 3). Furthermore, the

over-expression of the gene resulted in morphological changes

comparing to the control (Figs. 4A and 4B), such as the

obvi-ous pseudopod.

Results of Soft Agar Growth Assay

In the soft agar experiments, we first did one primary

experiment in a small number of samples. The results in the

experiment showed differences from each other in the different

groups. Then we increased the number of replicates in order to

avoid the experiment bias and did the experiments. The

67LR1-transfected 16HBE cells showed anchorage-independent

growth in semisolid medium (Fig. 4D) and also formed

pseu-dopod in soft agar compared with 16HBE (Fig. 4A) and

transfected eTEF1a1 gene control (Fig. 4C). The average

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results ± SD of the experiments are reported in Table 1.

Statistical analysis revealed a significant increase ( p < 0.001)

in 67LR1-transfected 16HBE cells compared with the two

negative control groups.

DISCUSSION

The identification and characterization of genes that are

differentially expressed during carcinogenesis or exposure to

carcinogens provide important information with regard to

understanding of the mechanisms responsible for malignant

transformation (Joseph et al., 2002). In the current

investiga-tion, we have used the 16HBE transformation system and SSH

analysis of gene expression to understand the molecular

mechanisms of carcinogenesis induced by anti-BPDE.

Analysis of differentially expressed genes by SSH technique

demonstrated overexpression of the 67LR1 gene in 16HBE-C

cells compared with 16HBE cells. The 67LR1 gene was

reported to be frequently detected in both carcinoma and

stromal cells in solid tumors (Givant-Horwitz et al., 2003).

This might explain abundant clinical and experimental data

suggesting a key role for the 67LR1 gene in the interaction

between cancer cells and the basement membrane glycoprotein

during tumor invasion and metastasis (Magnifico et al., 1996).

Several studies have clearly demonstrated an increase in

67LR1 expression in tumors compared with normal tissues

and a correlation between 67LR1 expression, invasive

pheno-type of the tumor, and poor prognosis (Magnifico et al., 1999).

But the main characterization of the 67LR1 gene is not very

clear.

Both 16HBE cells transfected with the 67LR1 gene and

colonies formed in soft agar from such cells showed

morpho-logical changes compared to the control 16HBE cells. One of

these changes is the presence of pseudopods. Stabilization of

FIG. 4. (A) The human bronchial epithelial cells (3250). (B) 16HBE cells transformed with 67LR1 gene. Arrows demonstrate the pseudopods. (C) Colony formed in 16HBEþeTEF1a1 gene group in semisolid agar. The colony did not show morphological changes (such as pseudopods). (D) Colony formed in 16HBEþ 67LR1 gene group in semisolid agar. Arrow demonstrates the pseudopods.

FIG. 3. Transfection-mediated overexpression of 67LR1 gene in 16HBE cells. M: DNA marker. 1, 2, and 3 are the results of semiquantitative PCR of 16HBE group, 16HBEþ vector group, and 16HBE þ 67LR1 group, respectively.

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these pseudopods determines the directionality of cell

move-ment (Nabi, 1999) and may be related to cell adhesive ability

and cell spreading ability (Zhao et al., 2005). Our results

suggest that overexpression of 67LR1 gene could be a

bio-marker of malignant transformation. Overexpression of the

67LR1 gene could also predict tumor metastasis. Further work

needs to be done to substantiate this hypothesis. The

signifi-cance of increased message expression of 67LR1 gene awaits

confirmation of the gene product at the protein level.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.

oxfordjournals.org/.

ACKNOWLEDGMENT

This work was supported by the grants from the National Natural Science Foundation of China (No. 30271111) and National Key Basic Research and Development Program (2002CB512905).

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TABLE 1

The Frequency of Colony Formation in Semisolid Agar

Group Number of parallel culture dishes Number of colony formation X ± SD 16HBE 5 1.33 ± 0.516 16HBEþ vector 5 1.57 ± 0.535 16HBEþ 67LR1 gene 5 20.98 ± 1.966*