A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth
<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS <...
| Main Authors: | , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2009-06-01
|
| Series: | BMC Cancer |
| Online Access: | http://www.biomedcentral.com/1471-2407/9/198 |
| _version_ | 1811314253066403840 |
|---|---|
| author | Yanagihara Kazuyoshi Kashima Lisa Idogawa Masashi Suzuki Hiromu Sasaki Yasushi Maruyama Reo Akashi Hirofumi Aoki Fumio Toyota Minoru Mita Hiroaki Fujita Masahiro Hosokawa Masao Kusano Masanobu Sabau Sorin Tatsumi Haruyuki Imai Kohzoh Shinomura Yasuhisa Tokino Takashi |
| author_facet | Yanagihara Kazuyoshi Kashima Lisa Idogawa Masashi Suzuki Hiromu Sasaki Yasushi Maruyama Reo Akashi Hirofumi Aoki Fumio Toyota Minoru Mita Hiroaki Fujita Masahiro Hosokawa Masao Kusano Masanobu Sabau Sorin Tatsumi Haruyuki Imai Kohzoh Shinomura Yasuhisa Tokino Takashi |
| author_sort | Yanagihara Kazuyoshi |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p> |
| first_indexed | 2024-04-13T11:09:36Z |
| format | Article |
| id | doaj.art-209eafa77cde4bf780eaec7e55a15616 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-04-13T11:09:36Z |
| publishDate | 2009-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-209eafa77cde4bf780eaec7e55a156162022-12-22T02:49:10ZengBMCBMC Cancer1471-24072009-06-019119810.1186/1471-2407-9-198A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growthYanagihara KazuyoshiKashima LisaIdogawa MasashiSuzuki HiromuSasaki YasushiMaruyama ReoAkashi HirofumiAoki FumioToyota MinoruMita HiroakiFujita MasahiroHosokawa MasaoKusano MasanobuSabau SorinTatsumi HaruyukiImai KohzohShinomura YasuhisaTokino Takashi<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p>http://www.biomedcentral.com/1471-2407/9/198 |
| spellingShingle | Yanagihara Kazuyoshi Kashima Lisa Idogawa Masashi Suzuki Hiromu Sasaki Yasushi Maruyama Reo Akashi Hirofumi Aoki Fumio Toyota Minoru Mita Hiroaki Fujita Masahiro Hosokawa Masao Kusano Masanobu Sabau Sorin Tatsumi Haruyuki Imai Kohzoh Shinomura Yasuhisa Tokino Takashi A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth BMC Cancer |
| title | A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth |
| title_full | A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth |
| title_fullStr | A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth |
| title_full_unstemmed | A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth |
| title_short | A novel method, digital genome scanning detects <it>KRAS </it>gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth |
| title_sort | novel method digital genome scanning detects it kras it gene amplification in gastric cancers involvement of overexpressed wild type kras in downstream signaling and cancer cell growth |
| url | http://www.biomedcentral.com/1471-2407/9/198 |
| work_keys_str_mv | AT yanagiharakazuyoshi anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT kashimalisa anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT idogawamasashi anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT suzukihiromu anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT sasakiyasushi anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT maruyamareo anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT akashihirofumi anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT aokifumio anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT toyotaminoru anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT mitahiroaki anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT fujitamasahiro anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT hosokawamasao anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT kusanomasanobu anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT sabausorin anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT tatsumiharuyuki anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT imaikohzoh anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT shinomurayasuhisa anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT tokinotakashi anovelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT yanagiharakazuyoshi novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT kashimalisa novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT idogawamasashi novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT suzukihiromu novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT sasakiyasushi novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT maruyamareo novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT akashihirofumi novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT aokifumio novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT toyotaminoru novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT mitahiroaki novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT fujitamasahiro novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT hosokawamasao novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT kusanomasanobu novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT sabausorin novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT tatsumiharuyuki novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT imaikohzoh novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT shinomurayasuhisa novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth AT tokinotakashi novelmethoddigitalgenomescanningdetectsitkrasitgeneamplificationingastriccancersinvolvementofoverexpressedwildtypekrasindownstreamsignalingandcancercellgrowth |