Cancer
Letters,
62 (1992)
Elsevier Scientific Publishers
199
199 - 203 Ireland Ltd.
Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung Ken Satoh, Koh Narumi, Toshihiko Sakai, Tatsuya Abe, Toshiaki Kikuchi, Keiko Matsushima, Satoshi Sindoh and Masakichi Motomiya Department
of [nfernal
Medicine,
The Research
Institute
for Tuberculosis
and
Cancer,
Tohoku
University,
Sendai
980
(Japan) (Received 9 September 1991) (Revision received 15 November (Accepted 18 November 1991)
1991)
Summary
Introduction
Cell-adhesiue protein laminin and its specific receptor play an important role in the processes of cancer proliferation, inoasion and metastasis. In the present study, we cloned the cDNAs of the 67-IcDa laminin receptor both from a human lung cell line (lMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-/&alaminin receptor mRNA was higher in SBC3 than in IMR90.
Laminin, a high molecular weight adhesive protein, is a main component of basement membrane. Therefore, it is considered that laminin receptors also play an important role in cell adhesion to the extracellular matrix, especially in the mechanism of cancer metastasis [3,9]. The 67-kDa laminin receptor is one of the non-integrin family receptors and is thought to be more specific and affinitive to laminin rather than the receptors belonging tc, the integrin family [8]. The cDNAs of the receptor have already been cloned from a breast cancer cell line [ 111 and a colon cancer cell line [13]. However, no cloning from human lung cell/tissue has been reported yet. In the present report, we obtained cDNAs both from a lung cancer cell line and from a normal lung cell line and compared these nucleotide sequences with each other.
Keywords: laminin; receptor; cancer; cDNA
human; lung
Correspondence to: Ken Satoh, Department Medicine, The Research Institute for Tuberculosis Tohoku
University,
4-
0304.3835/92/$05.00 Printed and Published
1 Seiryo-machi,
Sendai
of Internal and Cancer, 980. Japan.
0 1992 Elsevier Scientific Publishers in Ireland
Materials
and Methods
Cells Human lung cancer cell line SBC3, human lung fibroblast cell line IMR90, and human colIreland Ltd
195
22,
242
259 275
291
Fis.1 (a) Schematic diagram of human 67-kDa laminin receptor cDNA. The full length cDNA map is drawn according to ref. 13. Some restriction enzyme cutting sites are also shown in this map: Hf, HinfI; Hd, Hindlll; Hc, HincII; St, StuI; Sh, Sphl; P, PstI. The boxed region indicates the translated portion. Clones pKN30 and pKN41 were derived from the IMR90 cDNA library and pKN38 and pKN40 from the SBC3 cDNA library. (b) Nucleotide sequence of 67-kDa laminin receptor cDNA cloned from humam lung cancer cell line SBC3. Predictad amino acid sequence is also shown under the line of nucleotide sequence. The numerals on both sides of the lines indicate the amino acid numbers corresponding with that of ref. 13. When compared with the sequence of cDNA from IMR90, three nucleotide substitutions are found (indicated by ‘) and they cause two changes of amino acid. The alternative sequences in IMR90 are as follows: ‘73Leu(TTG); *llGIy(GGA); 228GI~(CAG). Chou-Fasman analysis, however, reveals no difference in the secondary structure of the protein.
201
resulted was 1.0 kb and 106, respectively. The SBC3 cDNA library was screened with 32P-labeled probe pKN30. The cDNA library of IMR90 was also rescreened in the same manner.
on cancer cell line DLDl were generous gifts from Japanese Cancer Research Resources Bank (JCRB, Tokyo, Japan). The cell line SBC3 is classified as a small cell lung cancer (oat-cell type) and is already characterized in a previous study [6].
DNA sequencing and analyses The nucleotide sequence was determined for both strands by the method of Sanger [lo]. DNA analysis and Chou-Fasman analysis [2] were done using DNASIS program (Hitachi, Yokohama, Japan).
Cloning of 67-kDa laminin receptor cDNA First, a short fragment of cDNA (almost 300 bp in size) was obtained from the IMR90 cDNA library (Clontech, Palo Alto, CA) by polymerase chain reaction (Perkin-Elmer Cetus, Emeryville, CA), in which an insert of DNA from the cDNA library was used as a template and two synthetic oligonucleotides (24 mer each) as primers. The 24 mers were synthesized according to the preceding sequences data from human umbilical vein endothelial cells [ll] . This 3 ’ portion of cDNA from IMR90 was subcloned in a plasmid vector (designated pKN30, see Fig. la) and used as a probe for hybridization thereafter. A X gt 11 cDNA library was constructed from an SBC3 cell line in the ordinary manner [7]. The average insert size and clone number of the cDNA library which
123
4
-1.2 kb
5
Northern blot hybridization Total cellular RNAs (15 fig) were extracted from IMR90, SBC3 and DLDl cell lines using guanidine thiocyanate [l] and electrophoresed with formaldehyde-denaturing agarose gel. After transfer into nitrocellulose membrane filters, hybridization was carried out with radiolabeled pKN30 insert. Results After
several
screenings
of SBC3
cDNA
ci
Cl.8
kb
Fig. 2 Expressions of 67-kDa laminin receptor mRNA in human lung cell lines. Total cellular RNA (15 pg) was electrophoresed with formaldehyde-denaturing gel and transferred into a nitrocellulose filter. The filter was hybridized with radiolabeled probe of either 67-kDa laminin receptor cDNA(a) or human fl-actin cDNA(b). Lane 1 and 4: SBCS; lanes 2 and 5: IMR90; lanes 3 and 6: DLDl.
202
library, two different clones (pKN38 and pKN40) were obtained which overlapped each other to encompass a whole translated portion (Fig. la). A full-length cDNA (pKN41) was also cloned from the IMR90 cDNA library. Figure lb demonstrates the nucleotide sequence of cDNA cloned from SBC3. The numbers of amino acid sequence followed Yow’s study [13]. When compared with that from IMR90, three differences of nucleotide sequence were found, and are listed as follows: (1) 173Leu(TTA) in SBCS, ‘73Leu(TTG) in IMR90; (2) 211GI~(GAA) in SBC3, 211Gly(GGA) in IMR90; (3) 228Leu(CTG) in SBC3 228G1~(CAG) in IMR90. The first substitution of nucleotide caused no change of amino acid. The second and the third substitution introduced amino acid changes, although no alteration of the secondary structure of protein resulted when examined by the ChouFasman analysis [Z] As shown in Fig. 2, the expressions of 67kDa laminin receptor mRNAs were estimated by Northern analyses. There was no difference in the size of the transcript (1.2 kb) among SBC3 (lane l), IMR90 (lane 2), and a colon cancer cell line DLDl (lane 3). However the level of mRNA in SBC3 was two times higher than that in IMR90. Disscusion In the present study, we demonstrated for the first time a full-length cDNA of 67-kDa lamnin receptor from a human lung cancer cell line. Determination of the nucleotide sequence revealed no major alternation but three base differences in comparison with that of a normal lung cell line. One substitution caused no change of amino acid, whereas the other two were accompanied with changes of amino acid. As a result of Chou-Fasman analysis, however, no difference in the secondary structure of the protein was found between both cell lines. These sequences are nearly identical with the nucleotide sequence of cDNA cloned from the colon cancer cell line DLDl [13], of whose sequences are as follows: ‘73Leu(TTG), 211G1~(GAA) and 22sG1~(CAG).
The significance of the amino acid changes found in lung cancer in the present study has not yet been elucidated. In murine cells, multiple copies of the laminin receptor gene are demonstrated and some of these genes have a few base substitutions causing changes of amino acid [4]. From these results, Fernandez suggests that there is a strong selective pressure to maintain gene expression of laminin receptor in murine cells. It has been demonstrated that the increased amount of 67-kDa laminin receptor upregulates properties of malignant cells such as metastatic potential and rapid growth [3,5,12]. In the case of human colon carcinoma, this mRNA is about g-fold more abundant than in adjacent normal colonic epithelium [13]. Northern analysis in the present study shows that the level of mRNA is twice as high in lung cancer cells than in normal lung cells, as demonstrated in Fig. 2. It is also suggested that the level of 67-kDa laminin receptor expression is related to cell differentiation, as shown in human colon carcinoma [13]. Along this line our preliminary study, using surgically resected lung cancer tissues, demonstrated that the gene expression of 67-kDa laminin receptor in the small cell type had increased more than in other differentiated (adeno or squamous) cell types of lung cancer (data not shown). References Chirgwin, J.M., ter, W.J.
Przybyla A.E.,
(1978)
MacDonald,
R. J. and Rut-
Isolation of biologically active ribonucleic
acid from sources enriched in ribonuclease.
Biochemistry,
18, 5294 - 5299. Chou.
P.Y.,
and Fasman,
G.D.
(1978)
Prediction of the
secondary structure of proteins from their amino acid sequence,
Adv.
Enzymol.
Relat.
Areas
Mol.
Biol.,
47,
45 - 148. Cixe, V., Castronovo,
V., Shmookler,
Grigioni,
L.A.
S.F.,
Liotta,
B.M.,
and Sobel,
Garbisa, S.,
M.E.
(1991)
In-
creased expression of the laminin receptor in human colon cancer. J. Natl. Cancer Inst., 83, 29Fernandez, M.E.
M.-T.,
Castronovo,
36.
V., Rao, C.N.
member of a multicopy gene family. Biochem. Res. Commun., Hand,
and Sobel,
(1991) The high affinity murine laminin receptor is a
P.H..
L. (1985)
Biophys.
175, 84-90.
Thor, A., Schlom, J., Rao, C.N.
and Liotta
Expression of laminin receptor in normal and
203
6
carcinomatous human antibody. Cancer Res., Hiraki, S., Miyai, M., Y., Ozawa S., Ikeda.
tissues as defined by a monoclonal 45, 2713-2719. Seto, T., Tamura, T., Watanabe, H., Nakata, Y., Ohnoshi. T. and
10
quencing 11
Kimura, 1. (1982) Establishment of human continuous cell lines from squamous cell, adeno- and small-cell carcinoma of the lung and the results of heterotransplantation. Lung Cancer (in Japanese), 22, 53 - 58. 7
8 9
Huynh. T.V., Young. R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in hgtl0 and Xgt 11. In: DNA Cloning, Vol. 1, pp. 49-78, Edttor: D.M. Glover. IRL Press, Oxford-Washington DC. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell, 48, 549-554. Juliano, R.L. (1987) Membrane receptors for extracellular matrix macromolecules: relationship to cell adhesion and metastasis. Biochim. Biophys. Acta, 907, tumor 261-278,
Sanger,
F., Nicklen S. and Coulson with chain-terminating
A.R. (1977) DNA seinhibitors,
Proc.
Natl.
Acad. Sci. U.S.A., 74, 5463-5467. Wever, U.M., Liotta, L.A., Jaye, M., Ricca, G.A., Drohan, W.N., Claysmith, A.P., Rao, C.N.. Wirth, P.. Coligan, J.E., Albrechtsen, R., Mudryj, M. and Sobel, M.E. (1986) Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin. Proc. Natl. Acad. Sci. U.S.A.. 83. 7137-7141.
12
13
Werer, U.M., Taraboletti, G., Sobel, M.E., Albrechtsen. R. and Liotta, L.A. (1987) Role of laminin receptor in tumor cell migration. Cancer Res., 47, 5691- 5698. Yow, H., Wang. J.M., Dhen. H.S., Lee, C., Steele, G.D. and Chen, L.B. (1988) Increased mRNA expression of a laminin-binding protein in human colon carcinoma: complete sequence of a full-length cDNA encoding the protein, Proc. Natl. Acad. Sci. U.S.A., 85, 6394 - 6398.
Letters,
62 (1992)
Elsevier Scientific Publishers
199
199 - 203 Ireland Ltd.
Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung Ken Satoh, Koh Narumi, Toshihiko Sakai, Tatsuya Abe, Toshiaki Kikuchi, Keiko Matsushima, Satoshi Sindoh and Masakichi Motomiya Department
of [nfernal
Medicine,
The Research
Institute
for Tuberculosis
and
Cancer,
Tohoku
University,
Sendai
980
(Japan) (Received 9 September 1991) (Revision received 15 November (Accepted 18 November 1991)
1991)
Summary
Introduction
Cell-adhesiue protein laminin and its specific receptor play an important role in the processes of cancer proliferation, inoasion and metastasis. In the present study, we cloned the cDNAs of the 67-IcDa laminin receptor both from a human lung cell line (lMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-/&alaminin receptor mRNA was higher in SBC3 than in IMR90.
Laminin, a high molecular weight adhesive protein, is a main component of basement membrane. Therefore, it is considered that laminin receptors also play an important role in cell adhesion to the extracellular matrix, especially in the mechanism of cancer metastasis [3,9]. The 67-kDa laminin receptor is one of the non-integrin family receptors and is thought to be more specific and affinitive to laminin rather than the receptors belonging tc, the integrin family [8]. The cDNAs of the receptor have already been cloned from a breast cancer cell line [ 111 and a colon cancer cell line [13]. However, no cloning from human lung cell/tissue has been reported yet. In the present report, we obtained cDNAs both from a lung cancer cell line and from a normal lung cell line and compared these nucleotide sequences with each other.
Keywords: laminin; receptor; cancer; cDNA
human; lung
Correspondence to: Ken Satoh, Department Medicine, The Research Institute for Tuberculosis Tohoku
University,
4-
0304.3835/92/$05.00 Printed and Published
1 Seiryo-machi,
Sendai
of Internal and Cancer, 980. Japan.
0 1992 Elsevier Scientific Publishers in Ireland
Materials
and Methods
Cells Human lung cancer cell line SBC3, human lung fibroblast cell line IMR90, and human colIreland Ltd
195
22,
242
259 275
291
Fis.1 (a) Schematic diagram of human 67-kDa laminin receptor cDNA. The full length cDNA map is drawn according to ref. 13. Some restriction enzyme cutting sites are also shown in this map: Hf, HinfI; Hd, Hindlll; Hc, HincII; St, StuI; Sh, Sphl; P, PstI. The boxed region indicates the translated portion. Clones pKN30 and pKN41 were derived from the IMR90 cDNA library and pKN38 and pKN40 from the SBC3 cDNA library. (b) Nucleotide sequence of 67-kDa laminin receptor cDNA cloned from humam lung cancer cell line SBC3. Predictad amino acid sequence is also shown under the line of nucleotide sequence. The numerals on both sides of the lines indicate the amino acid numbers corresponding with that of ref. 13. When compared with the sequence of cDNA from IMR90, three nucleotide substitutions are found (indicated by ‘) and they cause two changes of amino acid. The alternative sequences in IMR90 are as follows: ‘73Leu(TTG); *llGIy(GGA); 228GI~(CAG). Chou-Fasman analysis, however, reveals no difference in the secondary structure of the protein.
201
resulted was 1.0 kb and 106, respectively. The SBC3 cDNA library was screened with 32P-labeled probe pKN30. The cDNA library of IMR90 was also rescreened in the same manner.
on cancer cell line DLDl were generous gifts from Japanese Cancer Research Resources Bank (JCRB, Tokyo, Japan). The cell line SBC3 is classified as a small cell lung cancer (oat-cell type) and is already characterized in a previous study [6].
DNA sequencing and analyses The nucleotide sequence was determined for both strands by the method of Sanger [lo]. DNA analysis and Chou-Fasman analysis [2] were done using DNASIS program (Hitachi, Yokohama, Japan).
Cloning of 67-kDa laminin receptor cDNA First, a short fragment of cDNA (almost 300 bp in size) was obtained from the IMR90 cDNA library (Clontech, Palo Alto, CA) by polymerase chain reaction (Perkin-Elmer Cetus, Emeryville, CA), in which an insert of DNA from the cDNA library was used as a template and two synthetic oligonucleotides (24 mer each) as primers. The 24 mers were synthesized according to the preceding sequences data from human umbilical vein endothelial cells [ll] . This 3 ’ portion of cDNA from IMR90 was subcloned in a plasmid vector (designated pKN30, see Fig. la) and used as a probe for hybridization thereafter. A X gt 11 cDNA library was constructed from an SBC3 cell line in the ordinary manner [7]. The average insert size and clone number of the cDNA library which
123
4
-1.2 kb
5
Northern blot hybridization Total cellular RNAs (15 fig) were extracted from IMR90, SBC3 and DLDl cell lines using guanidine thiocyanate [l] and electrophoresed with formaldehyde-denaturing agarose gel. After transfer into nitrocellulose membrane filters, hybridization was carried out with radiolabeled pKN30 insert. Results After
several
screenings
of SBC3
cDNA
ci
Cl.8
kb
Fig. 2 Expressions of 67-kDa laminin receptor mRNA in human lung cell lines. Total cellular RNA (15 pg) was electrophoresed with formaldehyde-denaturing gel and transferred into a nitrocellulose filter. The filter was hybridized with radiolabeled probe of either 67-kDa laminin receptor cDNA(a) or human fl-actin cDNA(b). Lane 1 and 4: SBCS; lanes 2 and 5: IMR90; lanes 3 and 6: DLDl.
202
library, two different clones (pKN38 and pKN40) were obtained which overlapped each other to encompass a whole translated portion (Fig. la). A full-length cDNA (pKN41) was also cloned from the IMR90 cDNA library. Figure lb demonstrates the nucleotide sequence of cDNA cloned from SBC3. The numbers of amino acid sequence followed Yow’s study [13]. When compared with that from IMR90, three differences of nucleotide sequence were found, and are listed as follows: (1) 173Leu(TTA) in SBCS, ‘73Leu(TTG) in IMR90; (2) 211GI~(GAA) in SBC3, 211Gly(GGA) in IMR90; (3) 228Leu(CTG) in SBC3 228G1~(CAG) in IMR90. The first substitution of nucleotide caused no change of amino acid. The second and the third substitution introduced amino acid changes, although no alteration of the secondary structure of protein resulted when examined by the ChouFasman analysis [Z] As shown in Fig. 2, the expressions of 67kDa laminin receptor mRNAs were estimated by Northern analyses. There was no difference in the size of the transcript (1.2 kb) among SBC3 (lane l), IMR90 (lane 2), and a colon cancer cell line DLDl (lane 3). However the level of mRNA in SBC3 was two times higher than that in IMR90. Disscusion In the present study, we demonstrated for the first time a full-length cDNA of 67-kDa lamnin receptor from a human lung cancer cell line. Determination of the nucleotide sequence revealed no major alternation but three base differences in comparison with that of a normal lung cell line. One substitution caused no change of amino acid, whereas the other two were accompanied with changes of amino acid. As a result of Chou-Fasman analysis, however, no difference in the secondary structure of the protein was found between both cell lines. These sequences are nearly identical with the nucleotide sequence of cDNA cloned from the colon cancer cell line DLDl [13], of whose sequences are as follows: ‘73Leu(TTG), 211G1~(GAA) and 22sG1~(CAG).
The significance of the amino acid changes found in lung cancer in the present study has not yet been elucidated. In murine cells, multiple copies of the laminin receptor gene are demonstrated and some of these genes have a few base substitutions causing changes of amino acid [4]. From these results, Fernandez suggests that there is a strong selective pressure to maintain gene expression of laminin receptor in murine cells. It has been demonstrated that the increased amount of 67-kDa laminin receptor upregulates properties of malignant cells such as metastatic potential and rapid growth [3,5,12]. In the case of human colon carcinoma, this mRNA is about g-fold more abundant than in adjacent normal colonic epithelium [13]. Northern analysis in the present study shows that the level of mRNA is twice as high in lung cancer cells than in normal lung cells, as demonstrated in Fig. 2. It is also suggested that the level of 67-kDa laminin receptor expression is related to cell differentiation, as shown in human colon carcinoma [13]. Along this line our preliminary study, using surgically resected lung cancer tissues, demonstrated that the gene expression of 67-kDa laminin receptor in the small cell type had increased more than in other differentiated (adeno or squamous) cell types of lung cancer (data not shown). References Chirgwin, J.M., ter, W.J.
Przybyla A.E.,
(1978)
MacDonald,
R. J. and Rut-
Isolation of biologically active ribonucleic
acid from sources enriched in ribonuclease.
Biochemistry,
18, 5294 - 5299. Chou.
P.Y.,
and Fasman,
G.D.
(1978)
Prediction of the
secondary structure of proteins from their amino acid sequence,
Adv.
Enzymol.
Relat.
Areas
Mol.
Biol.,
47,
45 - 148. Cixe, V., Castronovo,
V., Shmookler,
Grigioni,
L.A.
S.F.,
Liotta,
B.M.,
and Sobel,
Garbisa, S.,
M.E.
(1991)
In-
creased expression of the laminin receptor in human colon cancer. J. Natl. Cancer Inst., 83, 29Fernandez, M.E.
M.-T.,
Castronovo,
36.
V., Rao, C.N.
member of a multicopy gene family. Biochem. Res. Commun., Hand,
and Sobel,
(1991) The high affinity murine laminin receptor is a
P.H..
L. (1985)
Biophys.
175, 84-90.
Thor, A., Schlom, J., Rao, C.N.
and Liotta
Expression of laminin receptor in normal and
203
6
carcinomatous human antibody. Cancer Res., Hiraki, S., Miyai, M., Y., Ozawa S., Ikeda.
tissues as defined by a monoclonal 45, 2713-2719. Seto, T., Tamura, T., Watanabe, H., Nakata, Y., Ohnoshi. T. and
10
quencing 11
Kimura, 1. (1982) Establishment of human continuous cell lines from squamous cell, adeno- and small-cell carcinoma of the lung and the results of heterotransplantation. Lung Cancer (in Japanese), 22, 53 - 58. 7
8 9
Huynh. T.V., Young. R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in hgtl0 and Xgt 11. In: DNA Cloning, Vol. 1, pp. 49-78, Edttor: D.M. Glover. IRL Press, Oxford-Washington DC. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell, 48, 549-554. Juliano, R.L. (1987) Membrane receptors for extracellular matrix macromolecules: relationship to cell adhesion and metastasis. Biochim. Biophys. Acta, 907, tumor 261-278,
Sanger,
F., Nicklen S. and Coulson with chain-terminating
A.R. (1977) DNA seinhibitors,
Proc.
Natl.
Acad. Sci. U.S.A., 74, 5463-5467. Wever, U.M., Liotta, L.A., Jaye, M., Ricca, G.A., Drohan, W.N., Claysmith, A.P., Rao, C.N.. Wirth, P.. Coligan, J.E., Albrechtsen, R., Mudryj, M. and Sobel, M.E. (1986) Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin. Proc. Natl. Acad. Sci. U.S.A.. 83. 7137-7141.
12
13
Werer, U.M., Taraboletti, G., Sobel, M.E., Albrechtsen. R. and Liotta, L.A. (1987) Role of laminin receptor in tumor cell migration. Cancer Res., 47, 5691- 5698. Yow, H., Wang. J.M., Dhen. H.S., Lee, C., Steele, G.D. and Chen, L.B. (1988) Increased mRNA expression of a laminin-binding protein in human colon carcinoma: complete sequence of a full-length cDNA encoding the protein, Proc. Natl. Acad. Sci. U.S.A., 85, 6394 - 6398.
