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Sequence analysis of the proximal promoter region of the human α-fetoprotein gene in hepatocellular carcinoma
CANCER LETTERS Cancer Letters 76 (1994) 93-99
Sequence
analysis of the proximal promoter region of the human a-fetoprotein gene in hepatocellular carcinoma
Kentarou Igarashi *, Yutaka Aoyagi, Showgo Ohkoshi, Tsuyoshi Yokota, Shigeki Mori, Tsuyoshi Suda, Tomoteru Kamimura, Hitoshi Asakura
(Received
12 May 1993; revision
received 4 November
1993: accepted
5 November
1992)
Abstract We have examined whether or not mutations exist in the proximal promoter region of the human ol-fctoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to IO 138 ngiml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP. nucleotides different from published ones were detected at -120 and - 113. These changes, however, probably reflect a DNA polymorphism. because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from -160 to -10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-I binding sites exist is not responsible for the re-expression of AFP in HCC. Key MYI&; Asymmetric
PCR: Direct sequencing;
Mutation;
1. Introduction
The a-fetoprotein (AFP) gene is physiologically expressed in the liver during the fetal period, and its expression becomes hardly detectable in adult * Corresponding author. 0304-3835/94/$06.00 0 I994 Elsevier Scientific SSDI 0304-3835(93)03227-V
Publishers
Ireland
Polymorphism
life [7,8]. However, when hepatocellular carcinoma (HCC) is formed, the AFP gene is re-expressed [l]. Thus, the mechanism of developmental regulation of the AFP gene and of its reactivation in HCC is interesting as a model of gene expression in ontogenesis and oncogenesis. AFP mRNA has been found to be increased in the HCC tissue of a Ltd. All rights reserved
94
blood leukocytes, DNA amplified (PCR).
patient with a high level of serum AFP [21]. This result, and the experiments in mice and rats [15,26]. indicate that the regulation of AFP synthesis occurs mainly at the level of transcription. In the 5’ flanking region of the human AFP gene, several c&-acting DNA sequences, such as the promoter. enhancer, silencer and glucocorticoid responsive element (GRE) have been detected [16,17,27], and some transcription factors that interact with these regulatory DNA sequences have been identified [13.25]. In addition, a study using transgenic mice demonstrated that a region within the first kilobase of DNA upstream of the AFP gene is sufficient to direct postnatal repression of the AFP gene [4]. Moreover, hepatoma-specific nuclear protein, termed AFPl. can bind to the proximal promoter region from -169 to +29 [25]. In DNase I footprinting assays, murine regulatory proteins, termed NP-III and NP-IV, not detected in the fetal liver but present in the adult liver, bound to the proximal promoter region (up to -200) [31]. All these results indicate the importance of the proximal promoter region of the AFP gene in the regulation of gene expression. In HCC, some mutations, such as activation of proto-oncogene [5], loss of heterozygosity [2] and chromosome translocation [lo] have been reported. So it may be possible that the mutations in the promoter region influence postnatal repression of the AFP gene and induce transcription initiation in HCC. However, full analysis of that region of the AFP gene in HCC has not been reported. In this study we examined whether or not mutations exist in the proximal promoter region of the AFP gene in HCC tissue, compared with surrounding liver cirrhosis (LC) tissue and peripheral
Table 1 Summary Patient 1
2 3 4
of patients no.
by direct sequencing of genomic by polymerase chain reaction
2. Materials and methods 1.1. Specinlens Six specimens taken from four patients were examined (Table 1). These six specimens consisted of one HCC tissue. one HCC and its corresponding non-cancerous (cirrhosis) tissues, one LC tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. One HCC tissue was taken at surgery, and the remainder were at autopsy. The diagnosis of the specimens was determined histologically. -7.1. kmplijkation of‘ DNA (first PCR) Genomic DNA was extracted by a standard proteinase K/SDS/phenol chloroform procedure [23]. To amplify and sequence the proximal promoter region of the AFP gene, four oligonucleotides were synthesized (Fig. 1). PCR was carried out according to Kogan et al. [ 121 with a slight modification, namely, 100 ~1 of reaction mixture contained 1 pg of genomic DNA in 70 mM Tris-HCl (pH 8.8). 20 mM (NH&Sod, 20 mM MgClz, 1 mM dithiothreitol, 0.1% bovine serum albumin, 0.1% Triton X-100, each primer (1 and 4) at 1 PM. 1.5 mM each of the deoxyribonucleotide triphosphate (dATP, dCTP. TTP, dGTP) and 5 units of Tuq DNA polymerase ( Perkin-Elmer/Cetus, Norwalk, CT). The reaction was carried out for 35 cycles, and each cycle consisted of incubations for 1 min at 90°C for denaturation, 45 s at 65°C for annealing and 2 min at 72°C for primer extension.
and specimens Sex/age
Specimen”
Serum AFP (ngml)
Serum HBsAg
PI49 F/50 Ml40 M/40
HCC, LC (autopsyb) HCC (surgeryb), Leukocytes HCC (autopsyb) LC (autopsyb)
2749 10 138 16 <5
+ + + +
“HCC, hepatocellular carcinoma; LC, liver cirrhosis; bMethod by which specimens were obtained.
Leukocytes.
peripheral
blood
leukocytes.
K. Igarashi Ed al. /Cancer
Letr. 76 (1994)
X-l
95
93-99
x-2
Exon 1,
1,
1
-4 2
-3
*
1
I
-600
primer
-400
-200
+1
+200
1
5-
-TCTGCAACTTAGGGACAAGT-3-
-284
h -265
2
5-
-TGCCCCAAAGAGCTCTGTGT-3-
-183
~-164
3
5’# -TGGCAGTGGTGGAAGCACAA-3.
+22-
4
5-
t61-
-GATTCCACCCACTTCATGGT-3’
+3 +42
Fig. 1. Proximal promoter region of AFP gene and locations of the primers. Primer pair I and 4 was used for the first and second PCR. and primer pair 2 and 3 was used for direct sequencing. X-l and X-2 indicate repetitive sequences designated by Gibbs et al. [6]. When PCR is carried out with primer I and 4, primer 1 can hybridize to the nucleotides both from -284 to -265 and from -510 to -491.
The product was electrophoresed through 3% ultrapure agarose gel in Tris-acetate buffer. The DNA fragment of interest was electrophoretically collected onto DEAE-cellulose membrane (Schleicher and Schuell NA-45), eluted from the membrane, precipitated with ethanol and resolved in 20 ~1 of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA as described [ 121. 2.3. Asymmetric PCR (second PCR) To synthesize single-stranded DNA (ssDNA), asymmetric PCR was carried out [9,11], using DNA purified above (lo-20 ng). The condition of the reaction was the same as that of the first PCR except for concentrations of primers (1 PM primer 1 and 0.02 PM primer 4, or 1 PM primer 4 and 0.02 PM primer 1). The reaction product was filtrated Biomedicals, through SupprecTM -02 (TaKaRa Kyoto) membrane to remove unincorporated dNTPs and oligonucleotides, and the retentate was dried and resolved in 10 ~1 of water.
2.4. Direct DNA sequencing Sequencing was performed by the dideoxymediated chain termination method [24]. Seven ~1 of the ssDNA obtained above, 1 ~1 of internal primer (primer 2 with antisense strand DNA or primer 3 with sense strand DNA) and 2 ~1 of sequencing buffer (200 mM Tris-HCl, pH 7.51100 mM MgCl MgC12/250 mM NaCI) were mixed and annealed at 65°C for 2 min. To that solution (10 pl), 1 ~1 of 100 mM dithiothreitol, 2 ~1 of labeling mixture (1.5 FM dGTP, 1.5 PM dATP, 1.5 PM dTTP), 5 PCi of [a-32P]dCTP (1000 Ciimmol) and 2 units of Sequenase (United States Biochemical Corporation, Cleveland, OH) were added and reacted for 4 min at room temperature. Then, aliquots (3.5 ~1) of the reaction mixture were brought to each of four tubes that contained 2.5 ~1 of appropriate termination mixture (four dNTPs each at 80 PM with appropriate dideoxynucleotide at 8 PM) and incubated for 5 min at 37°C. The reaction was terminated by adding 4 ~1 of stop sol-
96
Ml2
571
bp
345
bp
Fig. 2. Agarose gel electrophorcsis of genomic DNA amplified by PCR using primer I and 4. Lanes I and 2 represent PCRampliiied DNA taken from liver cirrhosis and hepatocellular carcinoma tissues. respectively. of patient I. Fragments of 345 and 571 base pairs are seen in both specimens. and only the 34S-base-pair fragment was purified for sequencing. as described in Materials and methods. M. markers of pBR 322 digested with Hinfl.
to be sequenced, and the DNA sequence was determined in sense and antisense strand. A representative result of direct sequencing is shown in Fig. 3. In patient 2 (a high concentration of serum AFP), the HCC tissue showed the G to T transition at -120 in one allele and the G to T transition at -1 13 in both alleles. These transitions. however, were also detected in the peripheral blood leukocytes of the same patient. So, these changes probably reflect a DNA polymorphism but not somatic mutations. In the HCC tissue taken from patient 3 (a normal concentration of serum AFP) and the LC tissue taken from patient 4 (a normal concentration of serum AFP) the nucleotides of those sites were identical to published ones [6,22], namely G at -120 and G at - 113 (Fig. 3). Both HCC and surrounding LC tissues taken from patient 1 (a high concentration of serum AFP) also showed the identical nucleotides to published ones at those sites (data not shown). Other parts of nucleotide sequences from -160 to -10 were identical to published ones in all patients we have examined, including patient 2. who had different nucleotide sequences at two sites. 4. Discussion
ution (95% formamide/ mM EDTAIO.OS% bromphenol blue/O.OS%, xylene cyan01 FF). The samples were analysed by standard electrophoresis and autoradiography. 3. Results DNA fragments of 345 and 571 base pairs were amplified by the first PCR using primer 1 and 4 (Fig. 2). Since there is a repetitive sequence, designated X-l and X-2, in the 5’ flanking region [22] (Fig. 1), primer I is expected to hybridize not only to 20 nucleotides from -284 to -265 of antisense strand but also to those from -510 to -491 of that strand. So detection of the two fragments by PCR is compatible with that context. Considering the ability of direct sequencing. only the 345base-pair fragment was purifed and sequenced as described in Materials and methods. A minimum of about 150 bases (from - 160 to -10) were able
It is well established that there are two ways of sequencing to detect the mutations of a genomic DNA: cloning and direct sequencing methods. When the cloning method is used. several clones should be sequenced, since there are two alleles in the genome. On the other hand, the direct sequencing is able to detect two alleles simultaneously. So, we applied the latter method in this experiment. In addition, we used the first and second PCR, including asymmetric PCR [9.11], to purify the DNA of interest and produce ssDNA. By using an ssDNA template and a third internal primer in the sequencing reaction, the sequencing ladders were clear, and it was not necessary to label the end of the sequencing primer, as described in several reports in which double-stranded PCR products were used as template [3,28.29] A tissue-specific gene expression in eukaryotes is controlled by interactions between specific regulatory factors and &-acting DNA sequence
-G/T
GATC
Leukocytes
2)
c
C
G/T
GATC
WC
(patient
3)
-G
-G
GATC
LC
(patient
4)
-G
-G
Fig. 3. Representative nucleotide sequences of the sense strand of the proximal promoter region of the AFP gene in hepatocellular carcinoma tissue. Sequencing reaction was carried out by using primer 2. Hepatocellular carcinoma (HCC) tissue of patient 2 harbors G to T transition at - 120 in one allele and G to T transition at -I 13 in both alleles. The same transitions were seen in peripheral blood leukocytes (leukocytes) taken from the same patient. In HCC tissue of patient 3 and liver cirrhosis (LC) tissue of patient 4. nucleotide sequences were identical to the published ones, as indicated on the right side.
GATC
HCC
(patient
K. Ipmrshi
98
[14]. A mutation of the regulatory DNA sequence induced a pathological state in beta-thalassemia [20,28]. As for the AFP production in HCC, a transient expression analysis indicated that chloramphenicol acetyltransferase activity was increased by the mutation in the region from -146 to -13 1 of the mouse AFP gene [30]. In the present study we have examined the promoter region from - 160 to - 10 in HCC tissues taken from patients with various serum AFP concentrations, compared with non-cancerous tissues. No apparent point mutations or deletions were detected in this region, although the different nucleotide sequence at - 120 and - 113 in one HCC tissue and peripheral blood leukocytes due to DNA polymorphism were observed. So, from our study in the limited number of patients. reexpression of the human AFP gene in HCC is not caused by the mutation of the promoter region from -160 to -10 where GRE [17] and HNF-1 binding sites [13] exist. To resolve completely the problem about the relationship between the reexpression of the AFP gene and the mutation of the regulatory sequence. the 5 ‘ flanking region up to -5 kilobases, where the enhancer and the silencer reside, should be examined. In most HCCs, irrespective of causative factors (hepatitis B, virus infections, aflatoxin exposure, and so on), production of AFP occurs. Therefore, it is suggested that there are certain common mechanisms of re-expression of the AFP gene. Recently it has been suggested that developmental regulation of the rat AFP gene can be done by antagonism between glucocorticoid recepter and jiislj~n [32]. Transforming growth factor /3 1 [19] and c-Ha-ras [ 181 suppressed the activity of the promoter region of the AFP gene in HUH-~ cells. So, the modulation of the regulatory factors that are important in cell differentiation and/or cell proliferation may induce not only oncogenesis but also production of AFP in the liver. Further studies are required on both the transcriptioncontrolling region and the regulatory factors to clarify the mechanism of re-expression of the AFP gene.
6
IO
II
5. References I
Abelev,
G.I. (1968) Production
of embryonal
serum
01-
I6
et ul. / Cuncer Letr. 76
( 1994 i 93-99
globulin by hepatomas: review of experimental and clinical data. Cancer Res.. 28. 1344-1350. Buetow, K.H.. Murray, J.C.. Israel, J.L.. London. W.T.. Smith. M., Kew. M., Blanquet. V., Brechot. C.. Redeker. A. and Govindarajah. S. (1989) Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA, 86. 8852-8856. Burmer, G.C. and Loeb, L.A. (1989) Mutations in the KRAS.2 oncogene during progressive stage of human colon carcinoma. Proc. Natl. Acad. Sci. USA. 86. 2403-2407. Camper. S.A. and Tilghman. S.M. (1989) Postnatal repression of the oc-fetoprotein gene is enhancer independent. Genes Dev., 3. 537-546. Dejean. A.. Bougueleret. L., Grzeshik. K.H. and Tiollais. P. (1986) Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature, 322, 70-72. Gibbs. P.E.M., Zielinski. R., Boyd, C. and Dugaiczyk, A. (1987) Structure, polymorphism. and novel repeated DNA elements revealed by a complete sequence of the human a-fetoprotein gene. Biochemistry, 26, I332- 1343. Gitlin. D. and Boesman. M. (1966) Serum a-fetoprotein. albumin. and y G-protein in the human conceptus. J. Clin. Invest.. 45. 1826-1838. Gitlin. D. and Boesman, M. (1967) Sites of serum cyfetoprotein synthesis in the human and in the rat. J. Clin. Invest., 46. 1010-1016. Gyllensten, U.B. and Erlich, H.A. (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. USA, 85. 7652-7656. Hino. 0.. Shows. T.B. and Rogler, C.E. (1986) Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17:18 translocation. Proc. Natl. Acad. Sci. USA, 83. 8338-8342. Kadowaki, T.. Kadowaki. H. and Taylor, S.I. (1990) A non-sense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA. 87. 658-662. Kogan. SC., Dohery. M. and Gitshcier. J. (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences: application to hemophilia A. N. Engl. J. Med.. 317, 985-990. Mandel, D.B. and Crabtree. G.R. (1991) HNF-I, a member of a novel class of dimerizing homeodomain proteins. J. Biol. Chem.. 266. 677-680. Maniatis, T.. Goodbourn. S. and Fischer, J.A. (1987) Regulation of inducible and tissue-specific gene expression. Science, 236. l237- 1245. Nahn. J.-L.. Danan. J.-L., Poiret. M., Tratner. 1.. JoseEstanyol. M. and Sala-Trepat, J.M. (1987) The rat ofetoprotein and albumin genes. J. Biol. Chem.. 262, 12479-12487. Nakabayashi. H.. Hashimoto, T., Miyao. Y.. Tjong, K.-
K. Igarashi et al. / Cmcer
Lert. 76 (1994)
99
93-99
K., Chan. J. and Tamaoki. T. (1991) A positiondependent silencer plays a major role in repressing ofetoprotein expression in human hepatoma. Mol. Ceil. Biol.. I I, 5885-5893. 17 Nakabayashi, H., Watanabe. K., Saito, A.. Otsuru, A.. Sawadaishi, K. and Tamaoki. T. (1989) Transcriptional regulation of cu-fetoprotein expression by dexamethasone in human hepatoma cells. J. Biol. Chem., 264. 266-271. 18 Nakao, K., Lawless, D.. Ohe. Y.. Miyao, Y.. Nakabayashi. H.. Kamiya, H.. Miura. K., Ohtsuka, E. and Tamaoki, T. (1990) c-Ha-ras down regulates the ofetoprotein gene but not the albumin gene in human hepatoma cells. Mol. Cell. Biol., IO. 1461-1469. 19 Nakao, K.. Nakata. K.. Mitsuoka, S.. Ohtsuru. A., Ido. A.. Hatano. M., Sato. Y., Nakayama, T.. Shima, M.. Kusumoto. Y.. Koji. T.. Tamaoki. T. and Nagataki, S. (1991) Transforming growth factor p I differentially regulates cY-fetoprotein and albumin in HUH-~ human hepatoma cells. Biochem. Biophys. Res. Comm., 174. I294- 1299. 20 Orkin, S.H.. Kazazian. H.H. Jr.. Antonarakis. S.E.,‘Goff. S.C., Boehm, CD.. Sexton. J.P., Waber. P.G. and Giardra. P.J.V. (1982) Linkage of fl-thalassaemia mutations and P-globin gene polymorphisms with DNA polymorphisms in human @globin gene cluster. Nature, 296, 627-63 I, 21 Otsuru, A.. Nagataki, S.. Koji. T. and Tamaoki. T. (1988) Analysis of alphafetoprotein gene expression in hepatocellular carcinoma and liver cirrhosis by in situ hybridization Cancer, 62, 1105-I 112. 22 Sakai. M., Morinaga, T., Urano. Y., Watanabe, K.. Wegmann. T.G. and Tamaoki, T. (1985) The human cyfetoprotein gene: sequence organiation and the 5’ flanking region. J. Biol. Chem.. 260. 5055-5060. 23 Sambrook. J.. Fritsch, E.F. and Maniatis. T. (1989) Molecular Cloning, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. New York. 24 Sanger. F.. Nicklen. S. and Coulson A.R. (1977) DNA se-
25
26
27
28
29
30
31
32
quencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Sawadaishi, K., Morinaga, T. and Tamaoki, T. (1988) Interraction of a hepatoma-specific nuclear factor with transcription-regulatory sequences of the human cyfetoprotein and albumin genes. Mol. Cell. Biol.. 8. 5 179-5 187. Tilghman, S.M. and Belayew. A. (1982) Transcriptional control of the murine albumin/o-fetoprotein locus during developement. Proc. Nat) Acad. Sci. USA, 79, 5254-5257. Watanabe, K., Saito, A. and Tamaoki. T. (1987) Cellspecific enhancer activity in a far upstream region of the human cu-fetoprotein gene. J. Biol. Chem., 262. 48124818. Wong, C.. Dowling, C.E.. Saiki, R.K.. Higuchi. R.G.. Erlich. H.A. and Kazazian, H.H. Jr. Characterization of fl-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature, 330. 384-386. Yandell. D.W.. Campbell, T.A.. Dayton, S.H., Petersen, R.. Walton. D., Little, J.B.. McConkie-Resell. A.. Buckley. E.G. and Dryja. T.P. (1989) Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling. N. Engl. J. Med.. 321. 1689-1695. Zhang. D.-E.. Ge, X.. Rabek. J.P. and Papaconstantinou, J. (1991) Functional analysis of the Irons-acting factor binding sites of the mouse a-fetoprotein proximal promoter by site-directed mutagenesis. J. Biol. Chem., 266, 21179-21185. Zhang, D.-E.. Hoyt, P.R. and Papaconstantinou. J. (1990) Localization of DNA protein-binding sites in the proximal and distal promoter regions of the mouse ofetoprotein gene. J. Biol. Chem.. 265. 3382-3391. Zhang. X.-K., Dong, J.-M. and Chiu. J.-F. (1991) Regulation of o-fetoprotein gene expression by antagonism between AP-I and the glucocorticoid receptor at their overlapping binding site. J. Biol. Chem., 266. 824888254.
Sequence
analysis of the proximal promoter region of the human a-fetoprotein gene in hepatocellular carcinoma
Kentarou Igarashi *, Yutaka Aoyagi, Showgo Ohkoshi, Tsuyoshi Yokota, Shigeki Mori, Tsuyoshi Suda, Tomoteru Kamimura, Hitoshi Asakura
(Received
12 May 1993; revision
received 4 November
1993: accepted
5 November
1992)
Abstract We have examined whether or not mutations exist in the proximal promoter region of the human ol-fctoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to IO 138 ngiml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP. nucleotides different from published ones were detected at -120 and - 113. These changes, however, probably reflect a DNA polymorphism. because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from -160 to -10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-I binding sites exist is not responsible for the re-expression of AFP in HCC. Key MYI&; Asymmetric
PCR: Direct sequencing;
Mutation;
1. Introduction
The a-fetoprotein (AFP) gene is physiologically expressed in the liver during the fetal period, and its expression becomes hardly detectable in adult * Corresponding author. 0304-3835/94/$06.00 0 I994 Elsevier Scientific SSDI 0304-3835(93)03227-V
Publishers
Ireland
Polymorphism
life [7,8]. However, when hepatocellular carcinoma (HCC) is formed, the AFP gene is re-expressed [l]. Thus, the mechanism of developmental regulation of the AFP gene and of its reactivation in HCC is interesting as a model of gene expression in ontogenesis and oncogenesis. AFP mRNA has been found to be increased in the HCC tissue of a Ltd. All rights reserved
94
blood leukocytes, DNA amplified (PCR).
patient with a high level of serum AFP [21]. This result, and the experiments in mice and rats [15,26]. indicate that the regulation of AFP synthesis occurs mainly at the level of transcription. In the 5’ flanking region of the human AFP gene, several c&-acting DNA sequences, such as the promoter. enhancer, silencer and glucocorticoid responsive element (GRE) have been detected [16,17,27], and some transcription factors that interact with these regulatory DNA sequences have been identified [13.25]. In addition, a study using transgenic mice demonstrated that a region within the first kilobase of DNA upstream of the AFP gene is sufficient to direct postnatal repression of the AFP gene [4]. Moreover, hepatoma-specific nuclear protein, termed AFPl. can bind to the proximal promoter region from -169 to +29 [25]. In DNase I footprinting assays, murine regulatory proteins, termed NP-III and NP-IV, not detected in the fetal liver but present in the adult liver, bound to the proximal promoter region (up to -200) [31]. All these results indicate the importance of the proximal promoter region of the AFP gene in the regulation of gene expression. In HCC, some mutations, such as activation of proto-oncogene [5], loss of heterozygosity [2] and chromosome translocation [lo] have been reported. So it may be possible that the mutations in the promoter region influence postnatal repression of the AFP gene and induce transcription initiation in HCC. However, full analysis of that region of the AFP gene in HCC has not been reported. In this study we examined whether or not mutations exist in the proximal promoter region of the AFP gene in HCC tissue, compared with surrounding liver cirrhosis (LC) tissue and peripheral
Table 1 Summary Patient 1
2 3 4
of patients no.
by direct sequencing of genomic by polymerase chain reaction
2. Materials and methods 1.1. Specinlens Six specimens taken from four patients were examined (Table 1). These six specimens consisted of one HCC tissue. one HCC and its corresponding non-cancerous (cirrhosis) tissues, one LC tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. One HCC tissue was taken at surgery, and the remainder were at autopsy. The diagnosis of the specimens was determined histologically. -7.1. kmplijkation of‘ DNA (first PCR) Genomic DNA was extracted by a standard proteinase K/SDS/phenol chloroform procedure [23]. To amplify and sequence the proximal promoter region of the AFP gene, four oligonucleotides were synthesized (Fig. 1). PCR was carried out according to Kogan et al. [ 121 with a slight modification, namely, 100 ~1 of reaction mixture contained 1 pg of genomic DNA in 70 mM Tris-HCl (pH 8.8). 20 mM (NH&Sod, 20 mM MgClz, 1 mM dithiothreitol, 0.1% bovine serum albumin, 0.1% Triton X-100, each primer (1 and 4) at 1 PM. 1.5 mM each of the deoxyribonucleotide triphosphate (dATP, dCTP. TTP, dGTP) and 5 units of Tuq DNA polymerase ( Perkin-Elmer/Cetus, Norwalk, CT). The reaction was carried out for 35 cycles, and each cycle consisted of incubations for 1 min at 90°C for denaturation, 45 s at 65°C for annealing and 2 min at 72°C for primer extension.
and specimens Sex/age
Specimen”
Serum AFP (ngml)
Serum HBsAg
PI49 F/50 Ml40 M/40
HCC, LC (autopsyb) HCC (surgeryb), Leukocytes HCC (autopsyb) LC (autopsyb)
2749 10 138 16 <5
+ + + +
“HCC, hepatocellular carcinoma; LC, liver cirrhosis; bMethod by which specimens were obtained.
Leukocytes.
peripheral
blood
leukocytes.
K. Igarashi Ed al. /Cancer
Letr. 76 (1994)
X-l
95
93-99
x-2
Exon 1,
1,
1
-4 2
-3
*
1
I
-600
primer
-400
-200
+1
+200
1
5-
-TCTGCAACTTAGGGACAAGT-3-
-284
h -265
2
5-
-TGCCCCAAAGAGCTCTGTGT-3-
-183
~-164
3
5’# -TGGCAGTGGTGGAAGCACAA-3.
+22-
4
5-
t61-
-GATTCCACCCACTTCATGGT-3’
+3 +42
Fig. 1. Proximal promoter region of AFP gene and locations of the primers. Primer pair I and 4 was used for the first and second PCR. and primer pair 2 and 3 was used for direct sequencing. X-l and X-2 indicate repetitive sequences designated by Gibbs et al. [6]. When PCR is carried out with primer I and 4, primer 1 can hybridize to the nucleotides both from -284 to -265 and from -510 to -491.
The product was electrophoresed through 3% ultrapure agarose gel in Tris-acetate buffer. The DNA fragment of interest was electrophoretically collected onto DEAE-cellulose membrane (Schleicher and Schuell NA-45), eluted from the membrane, precipitated with ethanol and resolved in 20 ~1 of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA as described [ 121. 2.3. Asymmetric PCR (second PCR) To synthesize single-stranded DNA (ssDNA), asymmetric PCR was carried out [9,11], using DNA purified above (lo-20 ng). The condition of the reaction was the same as that of the first PCR except for concentrations of primers (1 PM primer 1 and 0.02 PM primer 4, or 1 PM primer 4 and 0.02 PM primer 1). The reaction product was filtrated Biomedicals, through SupprecTM -02 (TaKaRa Kyoto) membrane to remove unincorporated dNTPs and oligonucleotides, and the retentate was dried and resolved in 10 ~1 of water.
2.4. Direct DNA sequencing Sequencing was performed by the dideoxymediated chain termination method [24]. Seven ~1 of the ssDNA obtained above, 1 ~1 of internal primer (primer 2 with antisense strand DNA or primer 3 with sense strand DNA) and 2 ~1 of sequencing buffer (200 mM Tris-HCl, pH 7.51100 mM MgCl MgC12/250 mM NaCI) were mixed and annealed at 65°C for 2 min. To that solution (10 pl), 1 ~1 of 100 mM dithiothreitol, 2 ~1 of labeling mixture (1.5 FM dGTP, 1.5 PM dATP, 1.5 PM dTTP), 5 PCi of [a-32P]dCTP (1000 Ciimmol) and 2 units of Sequenase (United States Biochemical Corporation, Cleveland, OH) were added and reacted for 4 min at room temperature. Then, aliquots (3.5 ~1) of the reaction mixture were brought to each of four tubes that contained 2.5 ~1 of appropriate termination mixture (four dNTPs each at 80 PM with appropriate dideoxynucleotide at 8 PM) and incubated for 5 min at 37°C. The reaction was terminated by adding 4 ~1 of stop sol-
96
Ml2
571
bp
345
bp
Fig. 2. Agarose gel electrophorcsis of genomic DNA amplified by PCR using primer I and 4. Lanes I and 2 represent PCRampliiied DNA taken from liver cirrhosis and hepatocellular carcinoma tissues. respectively. of patient I. Fragments of 345 and 571 base pairs are seen in both specimens. and only the 34S-base-pair fragment was purified for sequencing. as described in Materials and methods. M. markers of pBR 322 digested with Hinfl.
to be sequenced, and the DNA sequence was determined in sense and antisense strand. A representative result of direct sequencing is shown in Fig. 3. In patient 2 (a high concentration of serum AFP), the HCC tissue showed the G to T transition at -120 in one allele and the G to T transition at -1 13 in both alleles. These transitions. however, were also detected in the peripheral blood leukocytes of the same patient. So, these changes probably reflect a DNA polymorphism but not somatic mutations. In the HCC tissue taken from patient 3 (a normal concentration of serum AFP) and the LC tissue taken from patient 4 (a normal concentration of serum AFP) the nucleotides of those sites were identical to published ones [6,22], namely G at -120 and G at - 113 (Fig. 3). Both HCC and surrounding LC tissues taken from patient 1 (a high concentration of serum AFP) also showed the identical nucleotides to published ones at those sites (data not shown). Other parts of nucleotide sequences from -160 to -10 were identical to published ones in all patients we have examined, including patient 2. who had different nucleotide sequences at two sites. 4. Discussion
ution (95% formamide/ mM EDTAIO.OS% bromphenol blue/O.OS%, xylene cyan01 FF). The samples were analysed by standard electrophoresis and autoradiography. 3. Results DNA fragments of 345 and 571 base pairs were amplified by the first PCR using primer 1 and 4 (Fig. 2). Since there is a repetitive sequence, designated X-l and X-2, in the 5’ flanking region [22] (Fig. 1), primer I is expected to hybridize not only to 20 nucleotides from -284 to -265 of antisense strand but also to those from -510 to -491 of that strand. So detection of the two fragments by PCR is compatible with that context. Considering the ability of direct sequencing. only the 345base-pair fragment was purifed and sequenced as described in Materials and methods. A minimum of about 150 bases (from - 160 to -10) were able
It is well established that there are two ways of sequencing to detect the mutations of a genomic DNA: cloning and direct sequencing methods. When the cloning method is used. several clones should be sequenced, since there are two alleles in the genome. On the other hand, the direct sequencing is able to detect two alleles simultaneously. So, we applied the latter method in this experiment. In addition, we used the first and second PCR, including asymmetric PCR [9.11], to purify the DNA of interest and produce ssDNA. By using an ssDNA template and a third internal primer in the sequencing reaction, the sequencing ladders were clear, and it was not necessary to label the end of the sequencing primer, as described in several reports in which double-stranded PCR products were used as template [3,28.29] A tissue-specific gene expression in eukaryotes is controlled by interactions between specific regulatory factors and &-acting DNA sequence
-G/T
GATC
Leukocytes
2)
c
C
G/T
GATC
WC
(patient
3)
-G
-G
GATC
LC
(patient
4)
-G
-G
Fig. 3. Representative nucleotide sequences of the sense strand of the proximal promoter region of the AFP gene in hepatocellular carcinoma tissue. Sequencing reaction was carried out by using primer 2. Hepatocellular carcinoma (HCC) tissue of patient 2 harbors G to T transition at - 120 in one allele and G to T transition at -I 13 in both alleles. The same transitions were seen in peripheral blood leukocytes (leukocytes) taken from the same patient. In HCC tissue of patient 3 and liver cirrhosis (LC) tissue of patient 4. nucleotide sequences were identical to the published ones, as indicated on the right side.
GATC
HCC
(patient
K. Ipmrshi
98
[14]. A mutation of the regulatory DNA sequence induced a pathological state in beta-thalassemia [20,28]. As for the AFP production in HCC, a transient expression analysis indicated that chloramphenicol acetyltransferase activity was increased by the mutation in the region from -146 to -13 1 of the mouse AFP gene [30]. In the present study we have examined the promoter region from - 160 to - 10 in HCC tissues taken from patients with various serum AFP concentrations, compared with non-cancerous tissues. No apparent point mutations or deletions were detected in this region, although the different nucleotide sequence at - 120 and - 113 in one HCC tissue and peripheral blood leukocytes due to DNA polymorphism were observed. So, from our study in the limited number of patients. reexpression of the human AFP gene in HCC is not caused by the mutation of the promoter region from -160 to -10 where GRE [17] and HNF-1 binding sites [13] exist. To resolve completely the problem about the relationship between the reexpression of the AFP gene and the mutation of the regulatory sequence. the 5 ‘ flanking region up to -5 kilobases, where the enhancer and the silencer reside, should be examined. In most HCCs, irrespective of causative factors (hepatitis B, virus infections, aflatoxin exposure, and so on), production of AFP occurs. Therefore, it is suggested that there are certain common mechanisms of re-expression of the AFP gene. Recently it has been suggested that developmental regulation of the rat AFP gene can be done by antagonism between glucocorticoid recepter and jiislj~n [32]. Transforming growth factor /3 1 [19] and c-Ha-ras [ 181 suppressed the activity of the promoter region of the AFP gene in HUH-~ cells. So, the modulation of the regulatory factors that are important in cell differentiation and/or cell proliferation may induce not only oncogenesis but also production of AFP in the liver. Further studies are required on both the transcriptioncontrolling region and the regulatory factors to clarify the mechanism of re-expression of the AFP gene.
6
IO
II
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