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No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese
Cancer Letters 150 (2000) 201±204 www.elsevier.com/locate/canlet
No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese Nancy M. Wang a, Kun-Tu Yeh b, Chan-Hai Tsai a, Shu-Jung Chen a, Jan-Gowth Chang a,* a
Division of Molecular Medicine, Department of Medical Research, China Medical College Hospital, Taichung, Taiwan b Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan Received 23 August 1999; received in revised form 2 November 1999; accepted 4 November 1999
Abstract The protein tyrosine kinase activity of c-src proto-oncogene product, pp60 c-src, is elevated in a number of human cancers, including colon cancer. Phosphorylation of human pp60 c-src carboxy-terminal tyrosine 530 suppresses its kinase activity. A recent report suggested that the risk of colon cancer is higher for those who carry a C ! T transition mutation on codon 531 (Gln-531 ! Amber-531) of src gene. This mutation caused a prematured translation termination and up-regulated the kinase activity. To examine whether this mutation could be a risk factor for colon carcinoma in the Chinese population, we used the same PCR-based assay to analyze src genotypes of 131 colon cancers and other various types of carcinoma. No mutation was detected in all specimens that were screened in this study. Thus, mutation at Gln-531 of src gene does not seem to be involved in the development of colon cancer in Chinese ethnicity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: src; Colon cancer; Polymerase chain reaction; Proto-oncogene protein pp60 c-src; Chinese
1. Introduction Src family tyrosin kinases participate in the regulation of cell adhesion, cell growth and differentiation [1]. C-src, the normal counterpart of Rous sarcoma virus (RSV) src gene, is one of the nine src-related tyrosine kinases that have been identi®ed [2,3]. The proto-oncogene product, pp60 c-src, is a membraneassociated tyrosine kinase and was thought to be a key components in signal transduction pathways that relay signals received at the cell membrane to the cytoplasm and nucleus [4,5]. The kinase activity of src family members is regulated by phosphorylation and dephosphorylation of a * Corresponding author. Tel.: 1 886-4-205-2121 (ext. 7144); fax: 1 886-4-203-3295. E-mail address: [email protected] (J.G. Chang)
highly conserved tyrosine residue located at the carboxyl-terminus [6], which interacts intramoleculy with the SH2/SH3 functional domain. Dephosphorylation of tyrosin 527 (Tyr-527) in chicken c-src [7,8], which is equivalent to Tyr-530 in human, caused a 10±20-fold increase in the kinase activity of pp60 csrc in vitro [9,10]. Src protein, which lacked this tyrosine due to mutation, exhibited elevated kinase activity in vivo and the ability to cause cellular transformation and tumor formation [4,11]. Therefore, it has been suggested that the transforming potential of pp60 c-src is suppressed by a high level of phosphorylation at Try-527 in vivo [12]. The potential to become transforming proteins as a result of regulatory defect has been implicated in the development of human breast, liver and colon cancer [13±15]. Recently Irby et al. [16] had identi®ed a truncated mutation in human src, which is a C ! T transition
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00398-5
202
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
mutation on codon 531 (Glutamine ! Amber) could lead to tumorigenesis of colon cancer. In all the colon cancer patients they studied, 12% of the advanced colon cancer carried this mutation. This src531 mutation caused prematured translation termination and resulted in a truncated pp60 c-src protein. In recombinant src531 mutant, Tyr 530 was also phosphorylated; however, it was unable to function as a negative regulator [16]. To examine whether this src531 genetic alteration could represent a risk factor for colon carcinomas in the Chinese, we analyzed the src genotypes of colon cancer tissues, from both M0 and M1 stages, using the polymerase chain reaction (PCR)-based assay described by Irby's group [16]. Moreover, in this study, we also evaluated the potential associations between this src531 mutation and the risk of other types of carcinoma. 2. Materials and methods 2.1. Subjects and samples A hundred and thirty-one colon cancers, 53 gastric cancers, 94 intestine cancers, 18 esophageal cancers, 56 oral cancers, 60 breast cancers, 21 cervical cancers, 24 thyroid cancers, 22 kidney cancers, 13 dermatoma, 50 hepatoma, and 14 neuronal cancers were obtained from China Medical College Hospital and Changhua Christian Hospital. The tissues were frozen immediately after surgical resection and stored in liquid nitrogen until extraction of DNA. 2.2. DNA isolation and analysis of the src531 mutation DNA was extracted using a standard method as previously described [17]. The entire exon 12 region of src gene was PCR ampli®ed from the 100±500 ng genomic DNA using the following primers: sense, 5 0 ATGGTGAACCGCGAGGTGCT-3 0 ; and anti-sense, 5 0 -CGCCTGTGCCTAGAGGTTCT-3 0 . The ampli®cation conditions were as following: 35 cycles of denaturation at 948C for 2 min, annealing at 628C for 2 min, and extension at 728C for 3 min with a ®nal extension at 728C for 7 min. The PCR products were digested with ScaI restriction enzyme for differentiating normal and mutated allele. The digested
PCR products were then analyzed on a 3% agarose gel and visualized with ethidium bromide. 2.3. Sequencing analysis The PCR products were extracted using QIAquick PCR puri®cation kit (QIAEN, Inc., Valencia, CA) as described by the manufacture. The puri®ed products were sequenced using di-deoxy chain termination method as described in BigDyee Terminator Cycle Sequencing Kit (Perkin±Elmer Applied Biosystems, Inc., Foster City, CA) and the samples were analyzed by ABI PRISMe 310 Genetic Analyzer (Perkin± Elmer Applied Biosystems, Inc., Foster City, CA). 3. Result We analyzed DNA from 131 colon cancerous tissues, which include 115 early stage (M0) and 16 late stage (M1) specimens with distant metastases. The PCR reaction ampli®ed the potential mutation region in exon 12 of src gene and this 226-base-pair (bp) fragment (Fig. 1, lanes 1 and 3) from PCR was then digested with restriction enzyme ScaI. The C ! T mutation at condon 531 creates an ScaI site. Only the mutant allele would be cleaved, giving two fragments of 194 and 32 bp, whereas the normal allele is not cut and the fragment remains 226 bp long (Fig.1, lanes 2 and 4). Among 131 colon cancer tissues we screened by this method, none of the PCR product contained the SacI site. The PCR products were then sequenced and the results showed that all these PCR products had the normal exon 12 (Fig. 2). We also analyzed a large set of various carcinoma tissues using the same PCR method followed by direct sequencing (Table 1). No mutation was found in the src exon 12 of all the specimens we screened. 4. Discussion The previous study [16] reported that individuals who carry a C ! T mutation at condon 531 of src gene, may have genetic susceptibility to colon cancers. They also showed that protein product from this mutated gene had an increased kinase activity in vitro. In the present study, we investigated this
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
203
Fig. 2. Direct sequencing analysis of the possible mutagenic region in exon 12. The upper numbers indicating the corresponding amino acid sequence. The boxed area is the ScaI cutting site. Fig. 1. PCR-based analysis of src531 genetic alteration. Exon 12 of the src gene was ampli®ed by PCR which resulted in a 226-bp fragment (lane 1). After digesting with ScaI, the wild type was remained uncut (lanes 2±4) and the C ! T mutation would be cleaved into 194 and 32 bp fragments. Lanes 2 and 3, samples from the M0 and M1 stage colon cancerous tissues, respectively. Lane 4 is the control sample from a health individual. M, marker, 100 bp ladder.
hypothesis in the Chinese population using a similar PCR-based assay. However, our results do not con®rm those of the previous study. In our study, we found no genetic alteration in the entire exon 12 of src gene in either colon carcinoma or other types of cancerous tissues. Although colon carcinoma is one of the leading causes of cancer
death in Taiwan [18], it seems that this mutation does not related to any speci®c type of carcinoma in Chinese ethnicity. We believe that our ®nding con¯ict with those of Irby not because of difference in the sampling size, which was about the same. It is also unlikely that the differences between the studies could be attributed to difference in methods since we used similar PCR-based techniques. The fact that we detected no genetic abnormality in exon 12 of src, even in the M1 stage specimen, could be explained in several ways. First, this mutation might be relevant only for individuals from certain populations. The frequency of the src531 mutation may be varied according to ethnic group. The popula-
Table 1 Molecular screening of the Gln-531 in various cancerous tissues Cancer (no.)
Metastasis None
Colon (131) Gastric (53) Esophageal (18) Oral (56) Breast (60) Cervical (21) Thyroid (24) Kidney (22) Hepatoma (50) Dermatoma (13) Neuronal (14)
Yes (site)
Q 531
Amber 531
Q 531
Amber 531
115 54 18 52 58 21 24 22 50 13 14
0 0 0 0 0 0 0 0 0 0 0
16 (100% metastasis to the liver) 8 (lymph node) 0 4 (lymph node) 2 (lymph node) 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
204
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
tion in our study was predominantly Chinese and it is possible that the frequency of mutation is higher in Caucasian. Larger epidemiological studies should be undertaken among a variety of populations. Second, other environmental or genetic cofactors might be required or in¯uence the development of colon carcinogenesis. Finally, other mutations or polymorphic sites that are in linkage with Amber-531 to alter the kinase activity of pp60 c-src may exist.
[10]
Acknowledgements
[11]
This work was supported by a grant from China Medical College Hospital (DMR 89-052) to Dr N.M. Wang. References [1] J. Park, C.A. Cartwrigh, Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells, Mol. Cell. Biol. 15 (1995) 2374±2382. [2] J.B. Bolen, Non-receptor tyrosine kinase, Oncogene 8 (1993) 2025±2031. [3] C.E. Rudd, O. Janssen, K.V. Prasad, M. Raab, A. da Silva, J.C. Telfer, M. Yamamoto, Src-related protein tyrosin kinases and their surface receptors, Biochim. Biophys. Acta 1155 (1993) 239±266. [4] T.E. Kmieck, D. Shalloway, Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation, Cell 49 (1987) 75±82. [5] H. Piwnica-Worms, K.B. Saunders, T.M. Roberts, A.E. Smith, S.H. Cheng, Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src, Cell 49 (1987) 75± 82. [6] J.C. Williams, A. Weijland, S. Gon¯oni, A. Thompson, S.A. Ê Courtneidge, G. Superti-Furga, R.K. Wierenga, The 2.35 A crystal structure of the inactivated form of chicken src: a
[7] [8] [9]
[12] [13]
[14]
[15]
[16]
[17] [18]
dynamic molecule with multiple regulatory interactions, J. Mol. Biol. 274 (1997) 757±775. J.A. Cooper, K.L. Gould, C.A. Cartwright, T. Hunter, Tyr 527 is phosphorylatd in pp60c-src: implication for regulation, Science 231 (1986) 1431±1434. R. Jove, H. Hanafusa, Cell transformation by the viral src oncogene, Rev. Cell Biol. 3 (1987) 31±56. J.A. Cooper, C.S. King, Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src, Mol. Cell Biol. 6 (1986) 4467±4477. S.A. Courmeidge, Activation of pp60c-src kinase by middle T antigen binding or dephosphorylation, EMBO J. 4 (1985) 1471±1477. A. Tanaka, M. Salem, R.J. Eckroade, D.J. Fujita, Characterization of avian retroviruses carrying activated transforming human c-src genes and steps involved in expression of activated src-PKases in vitro, Oncogene Res. 5 (1990) 305±322. X. Zheng, Y. Wang, C.J. Pallen, Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase, Nature 359 (1992) 336±339. A.E. Ottenhoff-Kalff, G. Rijksen, E.A. van-Beurden, A. Hennipman, A.A. Michels, G.E. Staal, Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product, Cancer Res. 52 (1992) 4773±4778. M.S. Talamonti, M.S. Roh, S.A. Curley, G.E. Gallicck, Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer, J. Clin. Invest. 91 (1993) 53±60. T. Masaki, M. Okada, M. Tokuda, Y. Shiratori, O. Hatase, M. Shirai, M. Nishioka, M. Omata, Reduced C-terminal Src kinase (Csk) activities in hepatocelular carcinoma, Hepatology 29 (1999) 379±384. R.B. Irby, W. Mao, D. Coppola, J. Kang, J.M. Loubeau, W. Trudeau, R. Karl, D.J. Fujita, R. Jove, T.J. Yeatman, Activating SRC mutation in a subset of advanced human colon cancers, Nat. Genet. 21 (1999) 187±190. T.H. Su, J.C. Wang, H.H. Tseng, C.P. Chang, T.A. Chang, H.J. Wei, J.G. Chang, Analysis of FHIT transcripts is cervical and endometrial cancers, Int. J. Cancer 76 (1998) 216±222. General health statistics, in: Health and viral Statistics, Republic of China ROC Press, Taipei, 1998, pp. 86±108.
No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese Nancy M. Wang a, Kun-Tu Yeh b, Chan-Hai Tsai a, Shu-Jung Chen a, Jan-Gowth Chang a,* a
Division of Molecular Medicine, Department of Medical Research, China Medical College Hospital, Taichung, Taiwan b Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan Received 23 August 1999; received in revised form 2 November 1999; accepted 4 November 1999
Abstract The protein tyrosine kinase activity of c-src proto-oncogene product, pp60 c-src, is elevated in a number of human cancers, including colon cancer. Phosphorylation of human pp60 c-src carboxy-terminal tyrosine 530 suppresses its kinase activity. A recent report suggested that the risk of colon cancer is higher for those who carry a C ! T transition mutation on codon 531 (Gln-531 ! Amber-531) of src gene. This mutation caused a prematured translation termination and up-regulated the kinase activity. To examine whether this mutation could be a risk factor for colon carcinoma in the Chinese population, we used the same PCR-based assay to analyze src genotypes of 131 colon cancers and other various types of carcinoma. No mutation was detected in all specimens that were screened in this study. Thus, mutation at Gln-531 of src gene does not seem to be involved in the development of colon cancer in Chinese ethnicity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: src; Colon cancer; Polymerase chain reaction; Proto-oncogene protein pp60 c-src; Chinese
1. Introduction Src family tyrosin kinases participate in the regulation of cell adhesion, cell growth and differentiation [1]. C-src, the normal counterpart of Rous sarcoma virus (RSV) src gene, is one of the nine src-related tyrosine kinases that have been identi®ed [2,3]. The proto-oncogene product, pp60 c-src, is a membraneassociated tyrosine kinase and was thought to be a key components in signal transduction pathways that relay signals received at the cell membrane to the cytoplasm and nucleus [4,5]. The kinase activity of src family members is regulated by phosphorylation and dephosphorylation of a * Corresponding author. Tel.: 1 886-4-205-2121 (ext. 7144); fax: 1 886-4-203-3295. E-mail address: [email protected] (J.G. Chang)
highly conserved tyrosine residue located at the carboxyl-terminus [6], which interacts intramoleculy with the SH2/SH3 functional domain. Dephosphorylation of tyrosin 527 (Tyr-527) in chicken c-src [7,8], which is equivalent to Tyr-530 in human, caused a 10±20-fold increase in the kinase activity of pp60 csrc in vitro [9,10]. Src protein, which lacked this tyrosine due to mutation, exhibited elevated kinase activity in vivo and the ability to cause cellular transformation and tumor formation [4,11]. Therefore, it has been suggested that the transforming potential of pp60 c-src is suppressed by a high level of phosphorylation at Try-527 in vivo [12]. The potential to become transforming proteins as a result of regulatory defect has been implicated in the development of human breast, liver and colon cancer [13±15]. Recently Irby et al. [16] had identi®ed a truncated mutation in human src, which is a C ! T transition
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00398-5
202
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
mutation on codon 531 (Glutamine ! Amber) could lead to tumorigenesis of colon cancer. In all the colon cancer patients they studied, 12% of the advanced colon cancer carried this mutation. This src531 mutation caused prematured translation termination and resulted in a truncated pp60 c-src protein. In recombinant src531 mutant, Tyr 530 was also phosphorylated; however, it was unable to function as a negative regulator [16]. To examine whether this src531 genetic alteration could represent a risk factor for colon carcinomas in the Chinese, we analyzed the src genotypes of colon cancer tissues, from both M0 and M1 stages, using the polymerase chain reaction (PCR)-based assay described by Irby's group [16]. Moreover, in this study, we also evaluated the potential associations between this src531 mutation and the risk of other types of carcinoma. 2. Materials and methods 2.1. Subjects and samples A hundred and thirty-one colon cancers, 53 gastric cancers, 94 intestine cancers, 18 esophageal cancers, 56 oral cancers, 60 breast cancers, 21 cervical cancers, 24 thyroid cancers, 22 kidney cancers, 13 dermatoma, 50 hepatoma, and 14 neuronal cancers were obtained from China Medical College Hospital and Changhua Christian Hospital. The tissues were frozen immediately after surgical resection and stored in liquid nitrogen until extraction of DNA. 2.2. DNA isolation and analysis of the src531 mutation DNA was extracted using a standard method as previously described [17]. The entire exon 12 region of src gene was PCR ampli®ed from the 100±500 ng genomic DNA using the following primers: sense, 5 0 ATGGTGAACCGCGAGGTGCT-3 0 ; and anti-sense, 5 0 -CGCCTGTGCCTAGAGGTTCT-3 0 . The ampli®cation conditions were as following: 35 cycles of denaturation at 948C for 2 min, annealing at 628C for 2 min, and extension at 728C for 3 min with a ®nal extension at 728C for 7 min. The PCR products were digested with ScaI restriction enzyme for differentiating normal and mutated allele. The digested
PCR products were then analyzed on a 3% agarose gel and visualized with ethidium bromide. 2.3. Sequencing analysis The PCR products were extracted using QIAquick PCR puri®cation kit (QIAEN, Inc., Valencia, CA) as described by the manufacture. The puri®ed products were sequenced using di-deoxy chain termination method as described in BigDyee Terminator Cycle Sequencing Kit (Perkin±Elmer Applied Biosystems, Inc., Foster City, CA) and the samples were analyzed by ABI PRISMe 310 Genetic Analyzer (Perkin± Elmer Applied Biosystems, Inc., Foster City, CA). 3. Result We analyzed DNA from 131 colon cancerous tissues, which include 115 early stage (M0) and 16 late stage (M1) specimens with distant metastases. The PCR reaction ampli®ed the potential mutation region in exon 12 of src gene and this 226-base-pair (bp) fragment (Fig. 1, lanes 1 and 3) from PCR was then digested with restriction enzyme ScaI. The C ! T mutation at condon 531 creates an ScaI site. Only the mutant allele would be cleaved, giving two fragments of 194 and 32 bp, whereas the normal allele is not cut and the fragment remains 226 bp long (Fig.1, lanes 2 and 4). Among 131 colon cancer tissues we screened by this method, none of the PCR product contained the SacI site. The PCR products were then sequenced and the results showed that all these PCR products had the normal exon 12 (Fig. 2). We also analyzed a large set of various carcinoma tissues using the same PCR method followed by direct sequencing (Table 1). No mutation was found in the src exon 12 of all the specimens we screened. 4. Discussion The previous study [16] reported that individuals who carry a C ! T mutation at condon 531 of src gene, may have genetic susceptibility to colon cancers. They also showed that protein product from this mutated gene had an increased kinase activity in vitro. In the present study, we investigated this
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
203
Fig. 2. Direct sequencing analysis of the possible mutagenic region in exon 12. The upper numbers indicating the corresponding amino acid sequence. The boxed area is the ScaI cutting site. Fig. 1. PCR-based analysis of src531 genetic alteration. Exon 12 of the src gene was ampli®ed by PCR which resulted in a 226-bp fragment (lane 1). After digesting with ScaI, the wild type was remained uncut (lanes 2±4) and the C ! T mutation would be cleaved into 194 and 32 bp fragments. Lanes 2 and 3, samples from the M0 and M1 stage colon cancerous tissues, respectively. Lane 4 is the control sample from a health individual. M, marker, 100 bp ladder.
hypothesis in the Chinese population using a similar PCR-based assay. However, our results do not con®rm those of the previous study. In our study, we found no genetic alteration in the entire exon 12 of src gene in either colon carcinoma or other types of cancerous tissues. Although colon carcinoma is one of the leading causes of cancer
death in Taiwan [18], it seems that this mutation does not related to any speci®c type of carcinoma in Chinese ethnicity. We believe that our ®nding con¯ict with those of Irby not because of difference in the sampling size, which was about the same. It is also unlikely that the differences between the studies could be attributed to difference in methods since we used similar PCR-based techniques. The fact that we detected no genetic abnormality in exon 12 of src, even in the M1 stage specimen, could be explained in several ways. First, this mutation might be relevant only for individuals from certain populations. The frequency of the src531 mutation may be varied according to ethnic group. The popula-
Table 1 Molecular screening of the Gln-531 in various cancerous tissues Cancer (no.)
Metastasis None
Colon (131) Gastric (53) Esophageal (18) Oral (56) Breast (60) Cervical (21) Thyroid (24) Kidney (22) Hepatoma (50) Dermatoma (13) Neuronal (14)
Yes (site)
Q 531
Amber 531
Q 531
Amber 531
115 54 18 52 58 21 24 22 50 13 14
0 0 0 0 0 0 0 0 0 0 0
16 (100% metastasis to the liver) 8 (lymph node) 0 4 (lymph node) 2 (lymph node) 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
204
N.M. Wang et al. / Cancer Letters 150 (2000) 201±204
tion in our study was predominantly Chinese and it is possible that the frequency of mutation is higher in Caucasian. Larger epidemiological studies should be undertaken among a variety of populations. Second, other environmental or genetic cofactors might be required or in¯uence the development of colon carcinogenesis. Finally, other mutations or polymorphic sites that are in linkage with Amber-531 to alter the kinase activity of pp60 c-src may exist.
[10]
Acknowledgements
[11]
This work was supported by a grant from China Medical College Hospital (DMR 89-052) to Dr N.M. Wang. References [1] J. Park, C.A. Cartwrigh, Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells, Mol. Cell. Biol. 15 (1995) 2374±2382. [2] J.B. Bolen, Non-receptor tyrosine kinase, Oncogene 8 (1993) 2025±2031. [3] C.E. Rudd, O. Janssen, K.V. Prasad, M. Raab, A. da Silva, J.C. Telfer, M. Yamamoto, Src-related protein tyrosin kinases and their surface receptors, Biochim. Biophys. Acta 1155 (1993) 239±266. [4] T.E. Kmieck, D. Shalloway, Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation, Cell 49 (1987) 75±82. [5] H. Piwnica-Worms, K.B. Saunders, T.M. Roberts, A.E. Smith, S.H. Cheng, Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src, Cell 49 (1987) 75± 82. [6] J.C. Williams, A. Weijland, S. Gon¯oni, A. Thompson, S.A. Ê Courtneidge, G. Superti-Furga, R.K. Wierenga, The 2.35 A crystal structure of the inactivated form of chicken src: a
[7] [8] [9]
[12] [13]
[14]
[15]
[16]
[17] [18]
dynamic molecule with multiple regulatory interactions, J. Mol. Biol. 274 (1997) 757±775. J.A. Cooper, K.L. Gould, C.A. Cartwright, T. Hunter, Tyr 527 is phosphorylatd in pp60c-src: implication for regulation, Science 231 (1986) 1431±1434. R. Jove, H. Hanafusa, Cell transformation by the viral src oncogene, Rev. Cell Biol. 3 (1987) 31±56. J.A. Cooper, C.S. King, Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src, Mol. Cell Biol. 6 (1986) 4467±4477. S.A. Courmeidge, Activation of pp60c-src kinase by middle T antigen binding or dephosphorylation, EMBO J. 4 (1985) 1471±1477. A. Tanaka, M. Salem, R.J. Eckroade, D.J. Fujita, Characterization of avian retroviruses carrying activated transforming human c-src genes and steps involved in expression of activated src-PKases in vitro, Oncogene Res. 5 (1990) 305±322. X. Zheng, Y. Wang, C.J. Pallen, Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase, Nature 359 (1992) 336±339. A.E. Ottenhoff-Kalff, G. Rijksen, E.A. van-Beurden, A. Hennipman, A.A. Michels, G.E. Staal, Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product, Cancer Res. 52 (1992) 4773±4778. M.S. Talamonti, M.S. Roh, S.A. Curley, G.E. Gallicck, Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer, J. Clin. Invest. 91 (1993) 53±60. T. Masaki, M. Okada, M. Tokuda, Y. Shiratori, O. Hatase, M. Shirai, M. Nishioka, M. Omata, Reduced C-terminal Src kinase (Csk) activities in hepatocelular carcinoma, Hepatology 29 (1999) 379±384. R.B. Irby, W. Mao, D. Coppola, J. Kang, J.M. Loubeau, W. Trudeau, R. Karl, D.J. Fujita, R. Jove, T.J. Yeatman, Activating SRC mutation in a subset of advanced human colon cancers, Nat. Genet. 21 (1999) 187±190. T.H. Su, J.C. Wang, H.H. Tseng, C.P. Chang, T.A. Chang, H.J. Wei, J.G. Chang, Analysis of FHIT transcripts is cervical and endometrial cancers, Int. J. Cancer 76 (1998) 216±222. General health statistics, in: Health and viral Statistics, Republic of China ROC Press, Taipei, 1998, pp. 86±108.