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Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter
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Promoter paper
Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter Katsuyuki Hamada a; *, Hiroto Shinomiya b , Yoshihiro Asano b , Toshimasa Kihana a , Mari Iwamoto a , Yasushi Hanakawa c , Koji Hashimoto c , Susumu Hirose d , Satoru Kyo e , Masaharu Ito a a
Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan b Department of Bacteriology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan c Department of Dermatology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan d Department of Developmental Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan e Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Ishikawa 920-0934, Japan Received 23 October 2000; received in revised form 4 January 2001; accepted 8 January 2001
Abstract The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2, which are 95% identical in nucleotide sequence. SCCA1 is a papain-like cysteine proteinase inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor. We analyzed here the sequence and the promoter activity of the 5P-flanking region of the SCCA1 gene. Deletion analysis of SCCA1 and SCCA2 promoter identified a 471-bp core promoter region upstream of the transcription start site. The transcriptional activity of SCCA1 promoter was up-regulated in squamous cell carcinoma cells, compared with keratinocyte and adenocarcinoma cells. The ratios of SCCA1 to SCCA2 promoter activity in squamous cell carcinoma, keratinocyte and adenocarcinoma cells were respectively 1.6, 5.3 and 2.8. Position 350 of SCCA1 and SCCA2 promoters played an important role in determining the promoter activities of SCCA1 and SCCA2. These findings suggest that the transcriptional regulation of SCCA1 and SCCA2 might differ among squamous cell carcinoma, keratinocyte and adenocarcinoma cells, and that SCCA1 promoter might be a potential target of gene therapy for squamous cell carcinoma. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Squamous cell carcinoma antigen 1; Squamous cell carcinoma antigen 2; Luciferase assay; Squamous cell carcinoma ; Promoter ; Nucleotide sequence
Squamous cell carcinoma antigen (SCCA) is a circulating tumor marker for squamous cell carcinoma, especially that of cervix, head and neck, lung, and esophagus [1]. Elevated circulating levels of SCCA are not detected in patients with adenocarcinomas of the uterus, ovary, or breast [2]. Several studies show that increased serum SCCA levels are correlated with the extent of disease in patients with squamous cell carcinoma [2^4]. Higher SCCA levels are also indicative of deep tumor in¢ltration and lymph node involvement [5,6]. Moreover, the measurement of posttreatment SCCA levels is useful for monitoring the response to therapy and for predicting tumor recurrence and metastasis. Duk et al. [5] have found that
* Corresponding author. Fax: +81-899-60-5381; E-mail : [email protected]
79% of patients who failed treatment had persistent elevations in serum SCCA levels, whereas 91% of those without clinical evidence of tumor had undetectable SCCA. SCCA protein has been isolated from a metastatic, cervical squamous cell carcinoma [2]. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. The more telomeric gene, which corresponds to the original SCCA, is designated SCCA1, whereas the more centromeric gene is designated SCCA2. SCCA1 and SCCA2 are respectively 92% and 95% identical in amino acid and nucleotide sequences [7]. Although SCCA1 and SCCA2 are nearly identical members of the serpin superfamily, the signi¢cant di¡erences in their reactive site loops suggest that they inhibit di¡erent classes of proteinases. Biochemical analysis of recombinant proteins has shown that SCCA1 is a papain-like cysteine proteinase
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 1 7 4 - 9
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inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor [7]. These ¢ndings suggest that SCCA1 and SCCA2 may play important roles in controlling cell motility, invasiveness and proliferation of squamous cell carcinoma. Furthermore it is useful to determine the transcriptional regulatory elements of SCCA1 and SCCA2,
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since this might allow to use the promoters of these genes for tissue-speci¢c gene therapy of squamous cell carcinoma. A previous study has reported the cloning and characterization of the promoter region of SCCA2 [8]. However, the promoter region of SCCA1 remained to be identi¢ed.
Fig. 1. Nucleotide sequences of SCCA1 and SCCA2 promoter regions. The sequence at the top lines is SCCA1, and the sequence at the bottom lines is SCCA2. The transcription start site is indicated by the black dot on the sequence. The CATA box is underlined. Exon I and II are shown in the box. The 31 indicates the ¢rst nucleotide 5P to the start site of transcription.
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In the present study, we analyzed the promoter activity of the SCCA1 gene. Genomic DNA of SCCA1 was cloned, sequenced and compared with that of SCCA2. The 3.6-kb DNA fragment upstream of the SCCA1 gene was cloned into plasmids containing luciferase reporter gene, and DNA transfections were performed to test the promoter activity of cloned fragments. Subsequently, deletions were made in the cloned sequence to identify the regions essential for gene regulation and to determine the tissue-speci¢c transcriptional activities in squamous cell carcinoma cells, keratinocyte cells and adenocarcinoma cells. Human cervical squamous cell carcinoma SKGIIIa cell line was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). HEY was a gift from Dr. G. Mills, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA. Normal human keratinocyte K41, K42 and K50 cell lines were established by Dr. K. Hashimoto, Ehime University, Ehime, Japan. Human cervical squamous cell carcinoma HT-3, Ca Ski, MS751, ME-180 and C-33 A cell lines, human cervical adenocarcinoma HeLa cell line, human ovarian adenocarcinoma SK-OV-3 cell line, and human endometrial adenocarcinoma HEC-1-A and HEC-1-B cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in a humidi¢ed 5% CO2 /95% air incubator at 37³C. All cell lines except keratinocytes were grown in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum. Keratinocyte cell lines were grown in MCDD153 (Nissui Co., Tokyo, Japan) with bovine hypothalamus extract. The cDNA products from HT-3 were ampli¢ed by polymerase chain reaction (PCR), using common primers for exon 2 of SCCA1, yielding a 164-bp product. A human genomic library (EMBL3 SP6/T7, Clontech Laboratories Inc., Palo Alto, CA, USA) was screened with the 32 P-labeled PCR-ampli¢ed human SCCA1 partial DNA fragment. Ten positive clones were obtained after the second screening. Genomic DNA from exon 1 to the 3.7-kb fragment of the 5P-£anking region of SCCA1 was directly sequenced by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Foster, CA, USA). To compare SCCA1 and SCCA2 sequences, genomic DNA of SCCA2 was also cloned and sequenced by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). The transcription start site of SCCA1 mRNA was determined by a CapFinder method using a modi¢cation of the manufacturer's protocol (Clontech, Palo Alto, CA, USA) as shown in Fig. 2. A modi¢ed oligo(dT) primer primed the ¢rst-strand synthesis reaction. When reverse transcription (RT) reached the 5P-end of the mRNA, the enzyme's terminal transferase activity added a few additional nucleotides, primarily deoxycytidine, to the 3P-end of the cDNA. The 30-mer oligonucleotide, which had an oligo(G) sequence at its 3P-end, base-pairs with the deoxycytidine stretch, creating an extended template. RT then
Fig. 2. Determination of transcription start site by a CapFinder method. RNA from SKGIIIa cells was subjected to RT. The resulting fulllength, single-stranded cDNA contains the complete 5P-end of the mRNA, as well as sequences that are complementary to the oligonucleotide.
switched templates and continued replicating to the end of the oligonucleotide. The resulting full-length, singlestranded cDNA contained the complete 5P-end of the mRNA, as well as sequences that were complementary to the oligonucleotide. The sequence was determined by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). The nucleotide sequence data for the SCCA1 and SCCA2 genes reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequences with accession numbers AB034984 and AB035089, respectively. Various lengths of DNA fragments upstream from the exon 1 of SCCA1 and SCCA2 were PCR-ampli¢ed and inserted into the luciferase reporter vector PicaGene Basic, a promoterless and enhancerless vector (Toyo Ink MFG Co., Tokyo, Japan) in sense orientation relative to the luciferase coding sequence at MluI and BglII sites. The sequence of each insert was con¢rmed by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). Constructs containing SCCA1 and SCCA2 5P-£anking sequences and which were fused to the luciferase gene were transfected into cells in the presence of DOTAP liposomal transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA), according to the protocol recommended by the manufacturer. Brie£y, 1U105 cells seeded in a 12-well culture dish were exposed to transfection mixtures containing 1 Wg of luciferase reporter plasmids and 0.2 Wg of prenilla luciferase-herpes simplex virus thymidine kinase promoter control vector (Promega, Madison, WI, USA) at 37³C for 48 h. Dual luciferase assays were performed according to the manufacturer's protocol (Promega). Total RNA was isolated from cultured cells with RNA STAT 601 (TEL-TEST Inc., Houston, TX, USA). One Wg of total RNA diluted with DEPC-treated water was reverse-transcribed in a ¢nal volume of 20 Wl containing 5UFirst-Strand Synthesis Bu¡er (Life Technologies Inc., Gaithersburg, MD, USA), 10 mM dithiothreitol (DTT), 0.5 mM of each dNTP, 200 U of Moloney Murine Leukemia Virus Reverse Transcriptase (Life Technologies), and 0.5 Wg oligo(dT)12ÿ18 primer (Life Technologies) at 37³C for 60 min. The PCR was performed with human
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Fig. 3. Sequence analysis of the SCCA2 gene. Twelve kb of SCCA2 gene was sequenced by an ABI PRISM 310 Genetic Analyzer. Each exon was shown in the box. Numbers indicate the bases downstream (+) of the transcription start site. Numbers also indicate the bases at the start and end site of each exon.
SCCA1-, SCCA2-, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as an internal control standard)-speci¢c primers. The sequence of SCCA1 sense primer (SCCA 1-1) was 5P-AGGTAATATTGGCAGCAAT-3P, while that of antisense primer (SCCA 1-2) was 5P-GTAGGTGATGATCCGAATCC-3P. This primer set can detect cDNA encoding SCCA1 expressed as a 576-bp band. The sequence of SCCA2 sense primer (SCCA 2-1) was 5P-TGGGACTATTGGCAATGAT-3P, while that of antisense primer (SCCA 2-2) was 5P-GGAGATGATAATTCGACTAC-3P. This primer set can detect cDNA encoding SCCA2 expressed as a 576-bp band. The sequence of the GAPDH sense primer was 5P-CAAAGTTGTCATGGATGACC-3P, while that of antisense primer was 5P-CCATGGAGAAGGCTGGGG-3P, expressed as a 195-bp band. To the newly synthesized cDNA (1 Wl), a PCR reaction mixture containing 5 mM Tris^HCl pH 8.0, 10 mM NaCl, 10 WM EDTA, 0.1 mM DTT, 0.2 mM each dNTP, 2.5 mM MgCl2 , 0.01% Triton X-100, 0.5 U of Taq polymerase (Promega Corp., Madison, WI, USA), and 0.2 WM of primers was added to bring the ¢nal volume to 20 Wl. The PCR was heated to 94³C for 4 min and immediately cycled 30 times through a 30-s denaturing step at 94³C, a 30-s annealing step at 59³C, and a 60-s elongation step at 72³C. Aliquots of the PCR products were electrophoresed on 1% agarose gels (Life Technologies), and PCR fragments were visualized with ethidium bromide staining. To obtain the SCCA1 5P-£anking sequence, we isolated a genomic clone SCCA1 exons 1 and 2 and the 3.7-kb 5P£anking sequences of SCCA1 gene were isolated. Fig. 1 shows the nucleotide sequence of the 5P-£anking region of SCCA1. The transcription start site of SCCA1 was determined by a CapFinder method and set at +1 (Fig. 2). The putative TATA box motif (CATA box) was found at position 331 to 326. No canonical CCAAT box was found at the expected distance from the transcription start site. We also cloned and sequenced a genomic clone SCCA2, and its sequence and structure are shown in Figs. 1 and 3, respectively. Within the 500-bp 5P-£anking region, four nucleotides di¡ered between the SCCA1 and SCCA2 genes. The sequences of the W0.5-kb, 30.5W31.0-kb and 31.0W32.0-kb regions upstream from the SCCA1 and SCCA2 genes were respectively 99%, 98% and 95% identical, whereas those of the 32W33.7-kb regions upstream from the genes were 84% identical. To examine the transcriptional activities of SCCA1 and SCCA2 in squamous cell carcinoma, keratinocyte and ad-
enocarcinoma cells, a transient expression assay was performed. Luciferase reporter plasmids containing varying lengths of the 5P-£anking regions of the genes were constructed (pGV1-971, pGV1-721, pGV1-596, pGV1-471, pGV1-221 and pGV1-96 for SCCA1, and pGV2-971, pGV2-721, pGV2-596, pGV2-471, pGV2-221 and pGV296 for SCCA2) as shown in Fig. 4A and transfected into SKGIIIa, K42 and SK-OV-3 cells, and the cell lysates were tested in luciferase assays. Fig. 4B demonstrates the transcriptional activities in these three cell lines. Deletion analysis from 3971-bp to 3471-bp region upstream from the genes revealed gradual increase in transcriptional activity with decrease in length of promoters, suggesting the presence of sequence inhibiting activities of the genes between 3971 bp and 3596 bp. A 471-bp region of SCCA1 promoter (pGV1-471) demonstrated signi¢cant transcriptional activity in all cell lines. In particular, SKGIIIa cells conferred the highest transcriptional activity, at 94% of that of control reporter plasmid (pGV-Control) driven by SV40 enhancer/promoter. In contrast, the 471-bp region of SCCA2 promoter (pGV2-471) demonstrated signi¢cantly less transcriptional activity, at 52% of that of pGV1-471 in SKGIIIa cells. SK-OV-3 cells conferred the lowest transcriptional activity of pGV1-471, at 15% of that of control reporter plasmid. Furthermore, SK-OV-3 cells demonstrated lower transcriptional activity of pGV2-471 than SKGIIIa cells, at 31% of that of pGV1-471. K42 cells exhibited modest pGV1-471 transcriptional activity, equivalent to 33% of positive control activity, and the lowest pGV2-471 transcriptional activity, equivalent to 22% of that of pGV1-471. To determine whether SCCA1 or SCCA2 promoter is transcriptionally activated in each cell line, luciferase activities of the 471-bp SCCA1 promoter (pGV1-471) and 471-bp SCCA2 promoter (pGV2-471) constructs were compared. Fig. 4C demonstrates the ratio of transcriptional activity of pGV1-471 to that of pGV2-471 in squamous cell carcinoma, keratinocyte, and adenocarcinoma cells. Squamous cell carcinoma cells demonstrated the lowest ratio, 1.6 þ 0.2, while keratinocyte cells demonstrated the highest ratio, 5.3 þ 0.3. Adenocarcinoma cells demonstrated a modest ratio, 2.8 þ 0.6. To determine which sequence is responsible for the difference in transcriptional activities of the SCCA1 and SCCA2 core promoter plasmids (pGV1-471 and pGV2471), site-directed mutagenesis was performed as described previously [9]. Mutagenesis changed nucleotides 3348,
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3125, 350, or 323 in pGV1-471 to those in pGV2-471 (3348: TCG, 3125: GCA, 350: GCA, or 323: CCT) as shown in Fig. 5A. Nucleotide substitution at position 350 decreased the transcriptional activity of pGV1-471 to the level of pGV2-471 in SKGIIIa, K42 and SK-OV-3 cells (Fig. 5B). These results suggested
that the nucleotide at 350 is important for the transcriptional activity in the SCCA1 core promoter. To determine whether the SCCA1 gene or SCCA2 gene is more strongly expressed in squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines, cellular RNA samples from SKGIIIa, K42 and SK-OV-3 cells
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Fig. 4. (A) A schematic representation of reporter plasmids. One kb of 5P-truncated fragments of the promoter region upstream from SCCA1 and SCCA2 gene were inserted into luciferase (LUC) reporter vector in sense orientation. Arrow indicates the transcription start site. Numbers indicate the number of bases upstream (3) and downstream (+) of the transcription start site. The name of each reporter construct was assigned according to the 5P-end nucleotide numbers of inserted promoter sequences, upstream of the transcription start site. (B) Transcriptional activity of SCCA1 and SCCA2 promoter in various cell lines, and identi¢cation of core promoter region. Luciferase activity of reporter plasmids of SCCA1 and SCCA2 promoter was examined in cervical squamous cell carcinoma SKGIIIa cells, normal keratinocyte K42 cells, and ovarian adenocarcinoma SK-OV-3 cells. The plasmid (pGV-Control) driven by SV40 enhancer/promoter was used for positive control and pGV-Basic without enhancer/promoter was used for negative control. Luciferase activity in each plasmid was plotted as the ratio to the positive control plasmid (pGV-Control). Bars, S.D. (C) Transcriptional activities of 471-bp region of SCCA1 and SCCA2 promoter were compared among squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines. Ratios of SCCA1 to SCCA2 transcriptional activity were signi¢cantly di¡erent among squamous cell carcinoma, keratinocyte, and adenocarcinoma cells (P 6 0.001). Bars, S.D.
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were analyzed by RT-PCR. The SCCA1 gene was more strongly expressed in all three cell lines than the SCCA2 gene (Fig. 6). The SCCA1 and SCCA2 genes were the most strongly expressed in SKGIIIa cells. Ratios of SCCA1 to SCCA2 mRNA are similar to those of SCCA1 to SCCA2 promoter activities. In the present study, the highest activities of SCCA1
and SCCA2 promoters were found in the 471-bp region just upstream of these genes. It has been reported that a 0.4-kb fragment upstream of the SCCA2 gene exhibited signi¢cant promoter activity [8]. This is consistent with the ¢ndings of the present study, but the transcriptional activity of SCCA1 promoter remained to be determined. The present study is thus the ¢rst to report the sequence
Fig. 5. (A) A schematic representation of reporter plasmids. Site-directed mutagenesis was performed. Mutagenesis changed nucleotides 3348, 3125, 350, or 323 in pGV1-471 to those in pGV2-471 (3348: TCG, 3125: GCA, 350: GCA, or 323: CCT). (B) Transcriptional activities of 471-bp region of SCCA1 (pGV1-471), SCCA2 (pGV2-471), and SCCA1 promoter (pGV1-471-M-348, pGV1-471-M-125, pGV1-471-M-50, pGV1-471-M-23) mutated at position 3348, 3125, 350, or 323 were compared. Luciferase activity in each plasmid was plotted as the ratio to the positive control plasmid (pGV-Control). Bars, S.D.
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Fig. 6. PCR ampli¢cation of SCCA1 and SCCA2 cDNAs at 30 cycles with SCCA1- and SCCA2-speci¢c primer sets. PCR products were electrophoresed in 1% agarose gel. Total RNA from SKGIIIa, K42 and SK-OV-3 cells was reverse-transcribed and ampli¢ed by PCR. A GAPDH primer set was used as an internal control. PCR products were electrophoresed in 1% agarose gel.
analysis and transcriptional activity of SCCA1 promoter. The ratios of SCCA1 to SCCA2 promoter activity in squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines were respectively 1.6, 5.3 and 2.8; these ratios were similar to those of SCCA1 to SCCA2 mRNA expression in these three cell lines. These ¢ndings suggest that the transcriptional regulation of SCCA1 and SCCA2 might di¡er among squamous cell carcinoma, keratinocyte and adenocarcinoma cells. The SCCA1 promoter activity in SKGIIIa cells was three times that in K42 cells and six times that in SKOV-3 cells; these ratios were similar to the relative amounts of mRNA expression. SCCA1 promoter activity in SKGIIIa cells was almost the same as that of the SV40 promoter and higher than in K42 and SK-OV-3 cells. Rodriguez et al. [10] reported that an adenovirus variant containing minimal enhancer/promoter constructs derived from the 5P-£anking region of the human prostate-speci¢c antigen gene to drive the adenovirus type-5 E1A gene has a higher therapeutic index with a cell speci¢city for PSA (+) cells than for PSA (3) cells. Similarly, if the SCCA1 promoter was inserted into adenovirus to drive the adenovirus E1A gene, a higher therapeutic index with cell speci¢city could be obtained for squamous cell carcinomas
than for non-squamous cell carcinomas. Additionally, it has been reported that a novel two-step transcriptional activation system, in which the PSA promoter drives an arti¢cial transcriptional activator, GAL4-VP16 fusion protein, and this in turn activates transgene expression under the control of GAL4-responsive elements, greatly augmented transgene expression [11]. SCCA1 promoter can be used in place of PSA promoter in this two-step transcriptional activation system. The 471-bp region we tested of the SCCA1 promoter with highly speci¢c transcriptional activity for squamous cell carcinomas is thus quite promising for use in gene therapy of squamous cell carcinomas. At nucleotide position 350, the G residue in SCCA1 is replaced by the A residue in SCCA2. Site-directed mutagenesis from G to A at position 350 decreased the transcriptional activity of 471-bp SCCA1 promoter nearly to that of the corresponding region of SCCA2 promoter. The region surrounding the position 350 of SCCA2 promoter involves a binding site for Ikaros-1. Ikaros-1 was originally described as a transcriptional regulator present in activated lymphocytes that binds to a consensus DNA sequence within the regulatory elements of terminal deoxynucleotidyl transferase [12]. Ikaros proteins are generated
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from multiple, alternatively spliced mRNA species encoded by a single gene, and Ikaros-1 is present within the nucleus and is the most abundant isoform within the Ikaros family [13]. Ikaros-1 is found in normal infant bone marrow, and normal Ikaros-1 function is disrupted during leukemogenesis in infants with ALL [14]. Furthermore, the DNA-binding form of Ikaros-1 plays an important role in silencing of growth-regulatory genes in immature lymphocyte precursors [15]. The present results demonstrated that the transcriptional activity of SCCA2 promoter was less than that of SCCA1 promoter. Thus, Ikaros-1 might bind to position 350 of SCCA2 promoter and down-regulate SCCA2 gene expression. Alternatively, it is also possible that unknown transcription factors bind to position 350 of SCCA1 promoter and transactivate SCCA1 gene expression. This study was supported by a grant from the Ministry of Education, Science, Sports and Culture, Japan; and by a grant from the Ministry of Health and Welfare for a Comprehensive Strategy for Controlling Intractable Cancer, Japan. References [1] N. Takeshima, Y. Suminami, O. Takeda, H. Abe, N. Okuno, H. Kato, Expression of mRNA of SCC antigen in squamous cells, Tumor Biol. 13 (1992) 338^342. [2] H. Kato, H. Torigoe, Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma, Cancer 40 (1977) 1621^1628. [3] E.K. Senekjian, J.M. Young, P.A. Weiser, C.E. Spencer, S.E. Magic, A.L. Herbst, An evaluation of squamous cell carcinoma antigen in patients with cervical squamous cell carcinoma, Am. J. Obstet. Gynecol. 157 (1987) 433^439. [4] J.A. Bolli, D.L. Doering, J.R. Bosscher, T.G. Day, C.V. Rao, K. Owens, B. Kelly, J. Goldsmith, Squamous cell carcinoma antigen: clinical utility in squamous cell carcinoma of the uterine cervix, Gynecol. Oncol. 55 (1994) 169^173.
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[5] J.M. Duk, H.X.A. de Bruijn, K.H. Groenier, H. Hollema, K.A. ten Hoor, M. Krans, J.G. Aalders, Cancer of the uterine cervix: sensitivity and speci¢city of serum squamous cell carcinoma antigen determinations, Gynecol. Oncol. 39 (1990) 186^194. [6] N. Takeshima, Y. Hirai, K. Katase, K. Yano, K. Yamauchi, K. Hasumi, The value of squamous cell carcinoma antigen as a predictor of nodal metastasis in cervical cancer, Gynecol. Oncol. 68 (1998) 263^ 266. [7] S.S. Schneider, C. Schick, K.E. Fish, E. Miller, J.C. Pena, S.D. Treter, S.M. Hui, G.A. Silverman, A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene, Proc. Natl. Acad. Sci. USA 92 (1995) 3147^3151. [8] Y. Sakaguchi, F. Kishi, A. Murakami, Y. Suminami, H. Kato, Structural analysis of human SCC antigen 2 promoter, Biochim. Biophys. Acta 1444 (1999) 111^116. [9] R. Higuchi, Recombinant PCR, in: M.A. Innis (Ed.), PCR Protocols, Academic press, San Diego, CA, 1990, pp. 177^183. [10] R. Rodriguez, E.R. Schuur, H.Y. Lim, G.A. Henderson, J.W. Simons, D.R. Henderson, Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-speci¢c antigen-positive prostate cancer cells, Cancer Res. 57 (1997) 2559^ 2563. [11] T. Segawa, H. Takebayashi, Y. Kakehi, O. Yoshida, S. Narumiya, A. Kakizuka, Prostate-speci¢c ampli¢cation of expanded polyglutamine expression: a novel approach for cancer gene therapy, Cancer Res. 58 (1998) 2282^2287. [12] K. Lo, N.R. Landau, S.T. Smale, LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-speci¢c genes, Mol. Cell Biol. 11 (1991) 5229^5243. [13] A. Molnar, K. Georgopoulos, The Ikaros gene encodes a family of functionally diverse zinc ¢nger DNA-binding proteins, Mol. Cell Biol. 14 (1994) 8292^8303. [14] L. Sun, N. Heerema, L. Crotty, X. Wu, C. Navara, A. Vassilev, M. Sensel, G.H. Reaman, F.M. Uckun, Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia, Proc. Natl. Acad. Sci. USA 96 (1999) 680^685. [15] K.E. Brown, S.S. Guest, S.T. Smale, K. Hahm, M. Merkenschlager, A.G. Fisher, Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin, Cell 91 (1997) 845^854.
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Promoter paper
Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter Katsuyuki Hamada a; *, Hiroto Shinomiya b , Yoshihiro Asano b , Toshimasa Kihana a , Mari Iwamoto a , Yasushi Hanakawa c , Koji Hashimoto c , Susumu Hirose d , Satoru Kyo e , Masaharu Ito a a
Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan b Department of Bacteriology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan c Department of Dermatology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan d Department of Developmental Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan e Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Ishikawa 920-0934, Japan Received 23 October 2000; received in revised form 4 January 2001; accepted 8 January 2001
Abstract The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2, which are 95% identical in nucleotide sequence. SCCA1 is a papain-like cysteine proteinase inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor. We analyzed here the sequence and the promoter activity of the 5P-flanking region of the SCCA1 gene. Deletion analysis of SCCA1 and SCCA2 promoter identified a 471-bp core promoter region upstream of the transcription start site. The transcriptional activity of SCCA1 promoter was up-regulated in squamous cell carcinoma cells, compared with keratinocyte and adenocarcinoma cells. The ratios of SCCA1 to SCCA2 promoter activity in squamous cell carcinoma, keratinocyte and adenocarcinoma cells were respectively 1.6, 5.3 and 2.8. Position 350 of SCCA1 and SCCA2 promoters played an important role in determining the promoter activities of SCCA1 and SCCA2. These findings suggest that the transcriptional regulation of SCCA1 and SCCA2 might differ among squamous cell carcinoma, keratinocyte and adenocarcinoma cells, and that SCCA1 promoter might be a potential target of gene therapy for squamous cell carcinoma. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Squamous cell carcinoma antigen 1; Squamous cell carcinoma antigen 2; Luciferase assay; Squamous cell carcinoma ; Promoter ; Nucleotide sequence
Squamous cell carcinoma antigen (SCCA) is a circulating tumor marker for squamous cell carcinoma, especially that of cervix, head and neck, lung, and esophagus [1]. Elevated circulating levels of SCCA are not detected in patients with adenocarcinomas of the uterus, ovary, or breast [2]. Several studies show that increased serum SCCA levels are correlated with the extent of disease in patients with squamous cell carcinoma [2^4]. Higher SCCA levels are also indicative of deep tumor in¢ltration and lymph node involvement [5,6]. Moreover, the measurement of posttreatment SCCA levels is useful for monitoring the response to therapy and for predicting tumor recurrence and metastasis. Duk et al. [5] have found that
* Corresponding author. Fax: +81-899-60-5381; E-mail : [email protected]
79% of patients who failed treatment had persistent elevations in serum SCCA levels, whereas 91% of those without clinical evidence of tumor had undetectable SCCA. SCCA protein has been isolated from a metastatic, cervical squamous cell carcinoma [2]. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. The more telomeric gene, which corresponds to the original SCCA, is designated SCCA1, whereas the more centromeric gene is designated SCCA2. SCCA1 and SCCA2 are respectively 92% and 95% identical in amino acid and nucleotide sequences [7]. Although SCCA1 and SCCA2 are nearly identical members of the serpin superfamily, the signi¢cant di¡erences in their reactive site loops suggest that they inhibit di¡erent classes of proteinases. Biochemical analysis of recombinant proteins has shown that SCCA1 is a papain-like cysteine proteinase
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inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor [7]. These ¢ndings suggest that SCCA1 and SCCA2 may play important roles in controlling cell motility, invasiveness and proliferation of squamous cell carcinoma. Furthermore it is useful to determine the transcriptional regulatory elements of SCCA1 and SCCA2,
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since this might allow to use the promoters of these genes for tissue-speci¢c gene therapy of squamous cell carcinoma. A previous study has reported the cloning and characterization of the promoter region of SCCA2 [8]. However, the promoter region of SCCA1 remained to be identi¢ed.
Fig. 1. Nucleotide sequences of SCCA1 and SCCA2 promoter regions. The sequence at the top lines is SCCA1, and the sequence at the bottom lines is SCCA2. The transcription start site is indicated by the black dot on the sequence. The CATA box is underlined. Exon I and II are shown in the box. The 31 indicates the ¢rst nucleotide 5P to the start site of transcription.
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In the present study, we analyzed the promoter activity of the SCCA1 gene. Genomic DNA of SCCA1 was cloned, sequenced and compared with that of SCCA2. The 3.6-kb DNA fragment upstream of the SCCA1 gene was cloned into plasmids containing luciferase reporter gene, and DNA transfections were performed to test the promoter activity of cloned fragments. Subsequently, deletions were made in the cloned sequence to identify the regions essential for gene regulation and to determine the tissue-speci¢c transcriptional activities in squamous cell carcinoma cells, keratinocyte cells and adenocarcinoma cells. Human cervical squamous cell carcinoma SKGIIIa cell line was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). HEY was a gift from Dr. G. Mills, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA. Normal human keratinocyte K41, K42 and K50 cell lines were established by Dr. K. Hashimoto, Ehime University, Ehime, Japan. Human cervical squamous cell carcinoma HT-3, Ca Ski, MS751, ME-180 and C-33 A cell lines, human cervical adenocarcinoma HeLa cell line, human ovarian adenocarcinoma SK-OV-3 cell line, and human endometrial adenocarcinoma HEC-1-A and HEC-1-B cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in a humidi¢ed 5% CO2 /95% air incubator at 37³C. All cell lines except keratinocytes were grown in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum. Keratinocyte cell lines were grown in MCDD153 (Nissui Co., Tokyo, Japan) with bovine hypothalamus extract. The cDNA products from HT-3 were ampli¢ed by polymerase chain reaction (PCR), using common primers for exon 2 of SCCA1, yielding a 164-bp product. A human genomic library (EMBL3 SP6/T7, Clontech Laboratories Inc., Palo Alto, CA, USA) was screened with the 32 P-labeled PCR-ampli¢ed human SCCA1 partial DNA fragment. Ten positive clones were obtained after the second screening. Genomic DNA from exon 1 to the 3.7-kb fragment of the 5P-£anking region of SCCA1 was directly sequenced by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Foster, CA, USA). To compare SCCA1 and SCCA2 sequences, genomic DNA of SCCA2 was also cloned and sequenced by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). The transcription start site of SCCA1 mRNA was determined by a CapFinder method using a modi¢cation of the manufacturer's protocol (Clontech, Palo Alto, CA, USA) as shown in Fig. 2. A modi¢ed oligo(dT) primer primed the ¢rst-strand synthesis reaction. When reverse transcription (RT) reached the 5P-end of the mRNA, the enzyme's terminal transferase activity added a few additional nucleotides, primarily deoxycytidine, to the 3P-end of the cDNA. The 30-mer oligonucleotide, which had an oligo(G) sequence at its 3P-end, base-pairs with the deoxycytidine stretch, creating an extended template. RT then
Fig. 2. Determination of transcription start site by a CapFinder method. RNA from SKGIIIa cells was subjected to RT. The resulting fulllength, single-stranded cDNA contains the complete 5P-end of the mRNA, as well as sequences that are complementary to the oligonucleotide.
switched templates and continued replicating to the end of the oligonucleotide. The resulting full-length, singlestranded cDNA contained the complete 5P-end of the mRNA, as well as sequences that were complementary to the oligonucleotide. The sequence was determined by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). The nucleotide sequence data for the SCCA1 and SCCA2 genes reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequences with accession numbers AB034984 and AB035089, respectively. Various lengths of DNA fragments upstream from the exon 1 of SCCA1 and SCCA2 were PCR-ampli¢ed and inserted into the luciferase reporter vector PicaGene Basic, a promoterless and enhancerless vector (Toyo Ink MFG Co., Tokyo, Japan) in sense orientation relative to the luciferase coding sequence at MluI and BglII sites. The sequence of each insert was con¢rmed by an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). Constructs containing SCCA1 and SCCA2 5P-£anking sequences and which were fused to the luciferase gene were transfected into cells in the presence of DOTAP liposomal transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA), according to the protocol recommended by the manufacturer. Brie£y, 1U105 cells seeded in a 12-well culture dish were exposed to transfection mixtures containing 1 Wg of luciferase reporter plasmids and 0.2 Wg of prenilla luciferase-herpes simplex virus thymidine kinase promoter control vector (Promega, Madison, WI, USA) at 37³C for 48 h. Dual luciferase assays were performed according to the manufacturer's protocol (Promega). Total RNA was isolated from cultured cells with RNA STAT 601 (TEL-TEST Inc., Houston, TX, USA). One Wg of total RNA diluted with DEPC-treated water was reverse-transcribed in a ¢nal volume of 20 Wl containing 5UFirst-Strand Synthesis Bu¡er (Life Technologies Inc., Gaithersburg, MD, USA), 10 mM dithiothreitol (DTT), 0.5 mM of each dNTP, 200 U of Moloney Murine Leukemia Virus Reverse Transcriptase (Life Technologies), and 0.5 Wg oligo(dT)12ÿ18 primer (Life Technologies) at 37³C for 60 min. The PCR was performed with human
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Fig. 3. Sequence analysis of the SCCA2 gene. Twelve kb of SCCA2 gene was sequenced by an ABI PRISM 310 Genetic Analyzer. Each exon was shown in the box. Numbers indicate the bases downstream (+) of the transcription start site. Numbers also indicate the bases at the start and end site of each exon.
SCCA1-, SCCA2-, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as an internal control standard)-speci¢c primers. The sequence of SCCA1 sense primer (SCCA 1-1) was 5P-AGGTAATATTGGCAGCAAT-3P, while that of antisense primer (SCCA 1-2) was 5P-GTAGGTGATGATCCGAATCC-3P. This primer set can detect cDNA encoding SCCA1 expressed as a 576-bp band. The sequence of SCCA2 sense primer (SCCA 2-1) was 5P-TGGGACTATTGGCAATGAT-3P, while that of antisense primer (SCCA 2-2) was 5P-GGAGATGATAATTCGACTAC-3P. This primer set can detect cDNA encoding SCCA2 expressed as a 576-bp band. The sequence of the GAPDH sense primer was 5P-CAAAGTTGTCATGGATGACC-3P, while that of antisense primer was 5P-CCATGGAGAAGGCTGGGG-3P, expressed as a 195-bp band. To the newly synthesized cDNA (1 Wl), a PCR reaction mixture containing 5 mM Tris^HCl pH 8.0, 10 mM NaCl, 10 WM EDTA, 0.1 mM DTT, 0.2 mM each dNTP, 2.5 mM MgCl2 , 0.01% Triton X-100, 0.5 U of Taq polymerase (Promega Corp., Madison, WI, USA), and 0.2 WM of primers was added to bring the ¢nal volume to 20 Wl. The PCR was heated to 94³C for 4 min and immediately cycled 30 times through a 30-s denaturing step at 94³C, a 30-s annealing step at 59³C, and a 60-s elongation step at 72³C. Aliquots of the PCR products were electrophoresed on 1% agarose gels (Life Technologies), and PCR fragments were visualized with ethidium bromide staining. To obtain the SCCA1 5P-£anking sequence, we isolated a genomic clone SCCA1 exons 1 and 2 and the 3.7-kb 5P£anking sequences of SCCA1 gene were isolated. Fig. 1 shows the nucleotide sequence of the 5P-£anking region of SCCA1. The transcription start site of SCCA1 was determined by a CapFinder method and set at +1 (Fig. 2). The putative TATA box motif (CATA box) was found at position 331 to 326. No canonical CCAAT box was found at the expected distance from the transcription start site. We also cloned and sequenced a genomic clone SCCA2, and its sequence and structure are shown in Figs. 1 and 3, respectively. Within the 500-bp 5P-£anking region, four nucleotides di¡ered between the SCCA1 and SCCA2 genes. The sequences of the W0.5-kb, 30.5W31.0-kb and 31.0W32.0-kb regions upstream from the SCCA1 and SCCA2 genes were respectively 99%, 98% and 95% identical, whereas those of the 32W33.7-kb regions upstream from the genes were 84% identical. To examine the transcriptional activities of SCCA1 and SCCA2 in squamous cell carcinoma, keratinocyte and ad-
enocarcinoma cells, a transient expression assay was performed. Luciferase reporter plasmids containing varying lengths of the 5P-£anking regions of the genes were constructed (pGV1-971, pGV1-721, pGV1-596, pGV1-471, pGV1-221 and pGV1-96 for SCCA1, and pGV2-971, pGV2-721, pGV2-596, pGV2-471, pGV2-221 and pGV296 for SCCA2) as shown in Fig. 4A and transfected into SKGIIIa, K42 and SK-OV-3 cells, and the cell lysates were tested in luciferase assays. Fig. 4B demonstrates the transcriptional activities in these three cell lines. Deletion analysis from 3971-bp to 3471-bp region upstream from the genes revealed gradual increase in transcriptional activity with decrease in length of promoters, suggesting the presence of sequence inhibiting activities of the genes between 3971 bp and 3596 bp. A 471-bp region of SCCA1 promoter (pGV1-471) demonstrated signi¢cant transcriptional activity in all cell lines. In particular, SKGIIIa cells conferred the highest transcriptional activity, at 94% of that of control reporter plasmid (pGV-Control) driven by SV40 enhancer/promoter. In contrast, the 471-bp region of SCCA2 promoter (pGV2-471) demonstrated signi¢cantly less transcriptional activity, at 52% of that of pGV1-471 in SKGIIIa cells. SK-OV-3 cells conferred the lowest transcriptional activity of pGV1-471, at 15% of that of control reporter plasmid. Furthermore, SK-OV-3 cells demonstrated lower transcriptional activity of pGV2-471 than SKGIIIa cells, at 31% of that of pGV1-471. K42 cells exhibited modest pGV1-471 transcriptional activity, equivalent to 33% of positive control activity, and the lowest pGV2-471 transcriptional activity, equivalent to 22% of that of pGV1-471. To determine whether SCCA1 or SCCA2 promoter is transcriptionally activated in each cell line, luciferase activities of the 471-bp SCCA1 promoter (pGV1-471) and 471-bp SCCA2 promoter (pGV2-471) constructs were compared. Fig. 4C demonstrates the ratio of transcriptional activity of pGV1-471 to that of pGV2-471 in squamous cell carcinoma, keratinocyte, and adenocarcinoma cells. Squamous cell carcinoma cells demonstrated the lowest ratio, 1.6 þ 0.2, while keratinocyte cells demonstrated the highest ratio, 5.3 þ 0.3. Adenocarcinoma cells demonstrated a modest ratio, 2.8 þ 0.6. To determine which sequence is responsible for the difference in transcriptional activities of the SCCA1 and SCCA2 core promoter plasmids (pGV1-471 and pGV2471), site-directed mutagenesis was performed as described previously [9]. Mutagenesis changed nucleotides 3348,
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3125, 350, or 323 in pGV1-471 to those in pGV2-471 (3348: TCG, 3125: GCA, 350: GCA, or 323: CCT) as shown in Fig. 5A. Nucleotide substitution at position 350 decreased the transcriptional activity of pGV1-471 to the level of pGV2-471 in SKGIIIa, K42 and SK-OV-3 cells (Fig. 5B). These results suggested
that the nucleotide at 350 is important for the transcriptional activity in the SCCA1 core promoter. To determine whether the SCCA1 gene or SCCA2 gene is more strongly expressed in squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines, cellular RNA samples from SKGIIIa, K42 and SK-OV-3 cells
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Fig. 4. (A) A schematic representation of reporter plasmids. One kb of 5P-truncated fragments of the promoter region upstream from SCCA1 and SCCA2 gene were inserted into luciferase (LUC) reporter vector in sense orientation. Arrow indicates the transcription start site. Numbers indicate the number of bases upstream (3) and downstream (+) of the transcription start site. The name of each reporter construct was assigned according to the 5P-end nucleotide numbers of inserted promoter sequences, upstream of the transcription start site. (B) Transcriptional activity of SCCA1 and SCCA2 promoter in various cell lines, and identi¢cation of core promoter region. Luciferase activity of reporter plasmids of SCCA1 and SCCA2 promoter was examined in cervical squamous cell carcinoma SKGIIIa cells, normal keratinocyte K42 cells, and ovarian adenocarcinoma SK-OV-3 cells. The plasmid (pGV-Control) driven by SV40 enhancer/promoter was used for positive control and pGV-Basic without enhancer/promoter was used for negative control. Luciferase activity in each plasmid was plotted as the ratio to the positive control plasmid (pGV-Control). Bars, S.D. (C) Transcriptional activities of 471-bp region of SCCA1 and SCCA2 promoter were compared among squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines. Ratios of SCCA1 to SCCA2 transcriptional activity were signi¢cantly di¡erent among squamous cell carcinoma, keratinocyte, and adenocarcinoma cells (P 6 0.001). Bars, S.D.
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were analyzed by RT-PCR. The SCCA1 gene was more strongly expressed in all three cell lines than the SCCA2 gene (Fig. 6). The SCCA1 and SCCA2 genes were the most strongly expressed in SKGIIIa cells. Ratios of SCCA1 to SCCA2 mRNA are similar to those of SCCA1 to SCCA2 promoter activities. In the present study, the highest activities of SCCA1
and SCCA2 promoters were found in the 471-bp region just upstream of these genes. It has been reported that a 0.4-kb fragment upstream of the SCCA2 gene exhibited signi¢cant promoter activity [8]. This is consistent with the ¢ndings of the present study, but the transcriptional activity of SCCA1 promoter remained to be determined. The present study is thus the ¢rst to report the sequence
Fig. 5. (A) A schematic representation of reporter plasmids. Site-directed mutagenesis was performed. Mutagenesis changed nucleotides 3348, 3125, 350, or 323 in pGV1-471 to those in pGV2-471 (3348: TCG, 3125: GCA, 350: GCA, or 323: CCT). (B) Transcriptional activities of 471-bp region of SCCA1 (pGV1-471), SCCA2 (pGV2-471), and SCCA1 promoter (pGV1-471-M-348, pGV1-471-M-125, pGV1-471-M-50, pGV1-471-M-23) mutated at position 3348, 3125, 350, or 323 were compared. Luciferase activity in each plasmid was plotted as the ratio to the positive control plasmid (pGV-Control). Bars, S.D.
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Fig. 6. PCR ampli¢cation of SCCA1 and SCCA2 cDNAs at 30 cycles with SCCA1- and SCCA2-speci¢c primer sets. PCR products were electrophoresed in 1% agarose gel. Total RNA from SKGIIIa, K42 and SK-OV-3 cells was reverse-transcribed and ampli¢ed by PCR. A GAPDH primer set was used as an internal control. PCR products were electrophoresed in 1% agarose gel.
analysis and transcriptional activity of SCCA1 promoter. The ratios of SCCA1 to SCCA2 promoter activity in squamous cell carcinoma, keratinocyte and adenocarcinoma cell lines were respectively 1.6, 5.3 and 2.8; these ratios were similar to those of SCCA1 to SCCA2 mRNA expression in these three cell lines. These ¢ndings suggest that the transcriptional regulation of SCCA1 and SCCA2 might di¡er among squamous cell carcinoma, keratinocyte and adenocarcinoma cells. The SCCA1 promoter activity in SKGIIIa cells was three times that in K42 cells and six times that in SKOV-3 cells; these ratios were similar to the relative amounts of mRNA expression. SCCA1 promoter activity in SKGIIIa cells was almost the same as that of the SV40 promoter and higher than in K42 and SK-OV-3 cells. Rodriguez et al. [10] reported that an adenovirus variant containing minimal enhancer/promoter constructs derived from the 5P-£anking region of the human prostate-speci¢c antigen gene to drive the adenovirus type-5 E1A gene has a higher therapeutic index with a cell speci¢city for PSA (+) cells than for PSA (3) cells. Similarly, if the SCCA1 promoter was inserted into adenovirus to drive the adenovirus E1A gene, a higher therapeutic index with cell speci¢city could be obtained for squamous cell carcinomas
than for non-squamous cell carcinomas. Additionally, it has been reported that a novel two-step transcriptional activation system, in which the PSA promoter drives an arti¢cial transcriptional activator, GAL4-VP16 fusion protein, and this in turn activates transgene expression under the control of GAL4-responsive elements, greatly augmented transgene expression [11]. SCCA1 promoter can be used in place of PSA promoter in this two-step transcriptional activation system. The 471-bp region we tested of the SCCA1 promoter with highly speci¢c transcriptional activity for squamous cell carcinomas is thus quite promising for use in gene therapy of squamous cell carcinomas. At nucleotide position 350, the G residue in SCCA1 is replaced by the A residue in SCCA2. Site-directed mutagenesis from G to A at position 350 decreased the transcriptional activity of 471-bp SCCA1 promoter nearly to that of the corresponding region of SCCA2 promoter. The region surrounding the position 350 of SCCA2 promoter involves a binding site for Ikaros-1. Ikaros-1 was originally described as a transcriptional regulator present in activated lymphocytes that binds to a consensus DNA sequence within the regulatory elements of terminal deoxynucleotidyl transferase [12]. Ikaros proteins are generated
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from multiple, alternatively spliced mRNA species encoded by a single gene, and Ikaros-1 is present within the nucleus and is the most abundant isoform within the Ikaros family [13]. Ikaros-1 is found in normal infant bone marrow, and normal Ikaros-1 function is disrupted during leukemogenesis in infants with ALL [14]. Furthermore, the DNA-binding form of Ikaros-1 plays an important role in silencing of growth-regulatory genes in immature lymphocyte precursors [15]. The present results demonstrated that the transcriptional activity of SCCA2 promoter was less than that of SCCA1 promoter. Thus, Ikaros-1 might bind to position 350 of SCCA2 promoter and down-regulate SCCA2 gene expression. Alternatively, it is also possible that unknown transcription factors bind to position 350 of SCCA1 promoter and transactivate SCCA1 gene expression. This study was supported by a grant from the Ministry of Education, Science, Sports and Culture, Japan; and by a grant from the Ministry of Health and Welfare for a Comprehensive Strategy for Controlling Intractable Cancer, Japan. References [1] N. Takeshima, Y. Suminami, O. Takeda, H. Abe, N. Okuno, H. Kato, Expression of mRNA of SCC antigen in squamous cells, Tumor Biol. 13 (1992) 338^342. [2] H. Kato, H. Torigoe, Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma, Cancer 40 (1977) 1621^1628. [3] E.K. Senekjian, J.M. Young, P.A. Weiser, C.E. Spencer, S.E. Magic, A.L. Herbst, An evaluation of squamous cell carcinoma antigen in patients with cervical squamous cell carcinoma, Am. J. Obstet. Gynecol. 157 (1987) 433^439. [4] J.A. Bolli, D.L. Doering, J.R. Bosscher, T.G. Day, C.V. Rao, K. Owens, B. Kelly, J. Goldsmith, Squamous cell carcinoma antigen: clinical utility in squamous cell carcinoma of the uterine cervix, Gynecol. Oncol. 55 (1994) 169^173.
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[5] J.M. Duk, H.X.A. de Bruijn, K.H. Groenier, H. Hollema, K.A. ten Hoor, M. Krans, J.G. Aalders, Cancer of the uterine cervix: sensitivity and speci¢city of serum squamous cell carcinoma antigen determinations, Gynecol. Oncol. 39 (1990) 186^194. [6] N. Takeshima, Y. Hirai, K. Katase, K. Yano, K. Yamauchi, K. Hasumi, The value of squamous cell carcinoma antigen as a predictor of nodal metastasis in cervical cancer, Gynecol. Oncol. 68 (1998) 263^ 266. [7] S.S. Schneider, C. Schick, K.E. Fish, E. Miller, J.C. Pena, S.D. Treter, S.M. Hui, G.A. Silverman, A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene, Proc. Natl. Acad. Sci. USA 92 (1995) 3147^3151. [8] Y. Sakaguchi, F. Kishi, A. Murakami, Y. Suminami, H. Kato, Structural analysis of human SCC antigen 2 promoter, Biochim. Biophys. Acta 1444 (1999) 111^116. [9] R. Higuchi, Recombinant PCR, in: M.A. Innis (Ed.), PCR Protocols, Academic press, San Diego, CA, 1990, pp. 177^183. [10] R. Rodriguez, E.R. Schuur, H.Y. Lim, G.A. Henderson, J.W. Simons, D.R. Henderson, Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-speci¢c antigen-positive prostate cancer cells, Cancer Res. 57 (1997) 2559^ 2563. [11] T. Segawa, H. Takebayashi, Y. Kakehi, O. Yoshida, S. Narumiya, A. Kakizuka, Prostate-speci¢c ampli¢cation of expanded polyglutamine expression: a novel approach for cancer gene therapy, Cancer Res. 58 (1998) 2282^2287. [12] K. Lo, N.R. Landau, S.T. Smale, LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-speci¢c genes, Mol. Cell Biol. 11 (1991) 5229^5243. [13] A. Molnar, K. Georgopoulos, The Ikaros gene encodes a family of functionally diverse zinc ¢nger DNA-binding proteins, Mol. Cell Biol. 14 (1994) 8292^8303. [14] L. Sun, N. Heerema, L. Crotty, X. Wu, C. Navara, A. Vassilev, M. Sensel, G.H. Reaman, F.M. Uckun, Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia, Proc. Natl. Acad. Sci. USA 96 (1999) 680^685. [15] K.E. Brown, S.S. Guest, S.T. Smale, K. Hahm, M. Merkenschlager, A.G. Fisher, Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin, Cell 91 (1997) 845^854.
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