Vol.
168,
No.
April
30,
1990
2, 1990
BIOCHEMICAL
AND
RESEARCH
COMMUNICATIONS
Pages
cDNA CLONING
AND CHARACTERIZATION
HUMAN
PAPILLARY
Yukihito
Ishizaka,
THYROID
Carcinogenesis
February
Division,
Toshikazu
Ushijima,
l-l,
Tsukiji-5,
26,
1990
National
OF ret ACTIVATED
CARCINOMA
and Minako
Received
BIOPHYSICAL
IN A
CELL LINE*
Takashi
Sugimura
Nagao
Cancer Center
Chuo-ku.
402-408
Tokyo Japan,
Research
Institute
104
Summary: We obtained activated ret cDNAs (&TPC) from a human papillary thyroid carcinoma cell line, TPC- 1. and characterized its structure. The nucleotide sequence indicated that the recombination had occurred just upstream of the kinase domain of & proto-oncogene and that the position, where the conserved sequence of ret proto-oncogene starts in retTPC transcripts, was exactly the same as that of m-11 which we have pre=usly analyzed. Furthermore, a unique 13-glycine stretch, which is also present in a small subunit of the calcium dependent protease, calpain, was detected in the replaced sequence of ret TPC . The aberrant tyrosine kinase activity induced by the rearrangement of ret protooncogene could be involved in the development of papillary thyroid 019'30 Academic Press, Inc. carcinoma. The product of & proto-oncogene has been proposed to be one of the receptor-tyrosine kinases (1.2). Activated ret oncogene was first cloned from the NIH3T3 cell transformants (3,4). One of the activated forms, retII which we have characterized, had a rearrangement in the region just upstream of the kinase domain (5). However, this rearrangement was proved to have occurred during transfection (4). We have examined more than 100 tumors by Southern blot analysis for the presence of the activated ret in the original tumor DNAs, but none were found (data not shown). Recently, we found aberrant transcripts of ti proto-oncogene in a human papillary thyroid carcinoma cell line, TPC-1 (6.7). The sizes of the transcripts were about 2.0, 2.5, 4.0 and 5.0 kb (7). whereas the sizes of the major transcripts in neuroblastoma cell lines transcribed from non-altered *The nucleotide sequence data reported in this paper will appear in the DDBJ. EMBL and GenBank Nucleotide Sequence Databases under the accession number D90075 (B.B.R.C. 1990). 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
402
Vol.
168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a proto-oncogene were 3.9, 4.5, 6.0 and 7.0 kb (2). These transcripts in neuroblastoma were shown to be generated by alternative polyadenylation and/or alternative splicing in the 3’ region of I& proto-oncogene (2). Furthermore, the analysis of a-11 and I-& proto-oncogene cDNAs (2.5) indicated that two respective isoforms of oncogene products of a-11 or ret proto-oncogene differing in the carboxy-terminus would be encoded. The 9 carboxy-terminal amino acids of one of the products (9 amino acid type protein) were replaced with 51 different amino acids contained in the other product (51 amino acid type protein). Our extensive studies showed that each of the two smaller transcripts of the a-11 and ret protooncogene would encode 9 amino acid type protein (2.4). suggesting that those transcripts in TPC-1 would also encode the same type protein (7). The transcripts in TPC-1 hybridized to the ret cDNA fragment encompassing the kinase domain, but not to the cDNA fragments encoding transmembrane and extracellular domains (7). These data suggested that activated ret in TPC-1 would have a rearrangement in almost the same region as r&-II. In the present report, we describe the cDNA cloning and character&n-lion of the activated ret in TPC-1.
Materials
and Methods
cDNA Cloning. Poly(A)+ RNA was extracted from a human papillary thyroid carcinoma cell line, TPC-1 (6) using the fast track mRNA isolation kit (Invitronen, USA). A cDNA library was constructed according to the method described (8) with oligo(dT)12I8 as a primer and hgtl0 (Stratagene, USA) as a cloning vector. For screening the cDNA library, a 0.4 kb -RI-WI cDNA fragment encoding a 5’ part of the r& kinase domain was used as a probe (5). The cDNA fragment was labeled by a[azP]dCTP (Amersham, UK) using the multiprime DNA labeling system (Amersham, UK). After purification by plaque hybridization, the insert of each cDNA clone was su~lo~~dl to $e -RI site of Bluescript pSK-M13+ (Stratagene, USA). PO c eot e Hvbridization. Oligonucleotide was synthesized by 381A DNA synthesizer (Applied Biosystem. USA). Oligonucleotide was labeled by y132P]ATP (ICN. USA) using T4 polynucletide kinase (Toyobo, Japan). The kinase reaction and hybridization was performed under the condition reported (9). As a probe for detecting the cDNA clones encoding 9 amino acid type protein, the oligonucleotide (~‘CATCTAGTAGTXAATGCATGGGAC~‘) which had been used as the 9 amino acid specific probe (2.5). was used. Nucleotide Seauencina of the cDNA Clones and Structural Analysis of the Predicted Oncogene Product. The nucleotide sequencing was performed by chain termination method (10) using Sequenase (United States Biochemical, USA). As sequence primers, T3, T7 and an oligonucleotide (5’
168,
No.
April
30,
1990
2, 1990
BIOCHEMICAL
AND
RESEARCH
COMMUNICATIONS
Pages
cDNA CLONING
AND CHARACTERIZATION
HUMAN
PAPILLARY
Yukihito
Ishizaka,
THYROID
Carcinogenesis
February
Division,
Toshikazu
Ushijima,
l-l,
Tsukiji-5,
26,
1990
National
OF ret ACTIVATED
CARCINOMA
and Minako
Received
BIOPHYSICAL
IN A
CELL LINE*
Takashi
Sugimura
Nagao
Cancer Center
Chuo-ku.
402-408
Tokyo Japan,
Research
Institute
104
Summary: We obtained activated ret cDNAs (&TPC) from a human papillary thyroid carcinoma cell line, TPC- 1. and characterized its structure. The nucleotide sequence indicated that the recombination had occurred just upstream of the kinase domain of & proto-oncogene and that the position, where the conserved sequence of ret proto-oncogene starts in retTPC transcripts, was exactly the same as that of m-11 which we have pre=usly analyzed. Furthermore, a unique 13-glycine stretch, which is also present in a small subunit of the calcium dependent protease, calpain, was detected in the replaced sequence of ret TPC . The aberrant tyrosine kinase activity induced by the rearrangement of ret protooncogene could be involved in the development of papillary thyroid 019'30 Academic Press, Inc. carcinoma. The product of & proto-oncogene has been proposed to be one of the receptor-tyrosine kinases (1.2). Activated ret oncogene was first cloned from the NIH3T3 cell transformants (3,4). One of the activated forms, retII which we have characterized, had a rearrangement in the region just upstream of the kinase domain (5). However, this rearrangement was proved to have occurred during transfection (4). We have examined more than 100 tumors by Southern blot analysis for the presence of the activated ret in the original tumor DNAs, but none were found (data not shown). Recently, we found aberrant transcripts of ti proto-oncogene in a human papillary thyroid carcinoma cell line, TPC-1 (6.7). The sizes of the transcripts were about 2.0, 2.5, 4.0 and 5.0 kb (7). whereas the sizes of the major transcripts in neuroblastoma cell lines transcribed from non-altered *The nucleotide sequence data reported in this paper will appear in the DDBJ. EMBL and GenBank Nucleotide Sequence Databases under the accession number D90075 (B.B.R.C. 1990). 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
402
Vol.
168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a proto-oncogene were 3.9, 4.5, 6.0 and 7.0 kb (2). These transcripts in neuroblastoma were shown to be generated by alternative polyadenylation and/or alternative splicing in the 3’ region of I& proto-oncogene (2). Furthermore, the analysis of a-11 and I-& proto-oncogene cDNAs (2.5) indicated that two respective isoforms of oncogene products of a-11 or ret proto-oncogene differing in the carboxy-terminus would be encoded. The 9 carboxy-terminal amino acids of one of the products (9 amino acid type protein) were replaced with 51 different amino acids contained in the other product (51 amino acid type protein). Our extensive studies showed that each of the two smaller transcripts of the a-11 and ret protooncogene would encode 9 amino acid type protein (2.4). suggesting that those transcripts in TPC-1 would also encode the same type protein (7). The transcripts in TPC-1 hybridized to the ret cDNA fragment encompassing the kinase domain, but not to the cDNA fragments encoding transmembrane and extracellular domains (7). These data suggested that activated ret in TPC-1 would have a rearrangement in almost the same region as r&-II. In the present report, we describe the cDNA cloning and character&n-lion of the activated ret in TPC-1.
Materials
and Methods
cDNA Cloning. Poly(A)+ RNA was extracted from a human papillary thyroid carcinoma cell line, TPC-1 (6) using the fast track mRNA isolation kit (Invitronen, USA). A cDNA library was constructed according to the method described (8) with oligo(dT)12I8 as a primer and hgtl0 (Stratagene, USA) as a cloning vector. For screening the cDNA library, a 0.4 kb -RI-WI cDNA fragment encoding a 5’ part of the r& kinase domain was used as a probe (5). The cDNA fragment was labeled by a[azP]dCTP (Amersham, UK) using the multiprime DNA labeling system (Amersham, UK). After purification by plaque hybridization, the insert of each cDNA clone was su~lo~~dl to $e -RI site of Bluescript pSK-M13+ (Stratagene, USA). PO c eot e Hvbridization. Oligonucleotide was synthesized by 381A DNA synthesizer (Applied Biosystem. USA). Oligonucleotide was labeled by y132P]ATP (ICN. USA) using T4 polynucletide kinase (Toyobo, Japan). The kinase reaction and hybridization was performed under the condition reported (9). As a probe for detecting the cDNA clones encoding 9 amino acid type protein, the oligonucleotide (~‘CATCTAGTAGTXAATGCATGGGAC~‘) which had been used as the 9 amino acid specific probe (2.5). was used. Nucleotide Seauencina of the cDNA Clones and Structural Analysis of the Predicted Oncogene Product. The nucleotide sequencing was performed by chain termination method (10) using Sequenase (United States Biochemical, USA). As sequence primers, T3, T7 and an oligonucleotide (5’
