DHPLC分析肺癌患者Rb2/p130、p53基因突变及其临床意义
|
摘要
[目的]
1 DHPLC分析肺癌患者组织标本Rb2／p130和p53突变。
2 DHPLC分析肺癌患者痰标本Rb2／p130和p53突变及其作为临床分子诊断标记的可行性。
[方法]
1 基因组DNA提取自52例肺癌组织标本(44例NSCLC，8例SCLC)和47例痰标本(35例肺癌，12例对照)以及相应的外周血淋巴细胞。
2 PCR扩增Rb2／p130基因Exon19～22和p53基因Exon5～9。
3 DHPLC分析PCR扩增产物检测Rb2／p130和p53基因突变。
[结果]
1 组织标本Rb2／p130突变检出率23.08％(12／52)，其中腺癌32.00％(8／25)、鳞癌15.79％(3／19)、小细胞癌12.50％(1／8)，三者差异无显著意义(P＝0.403)。Exon21突变10例次，Exon20和Exon22突变各1例，未见Exon19突变。44例NSCLC Rb2／p130突变检出率与患者的性别、年龄、吸烟、病理类型、疾病分期等均无相关性(所有P＞0.05)。痰标本肺癌组Rb2／p130突变检出率22.86％(8／35)，对照组未检出一例，差异有显著意义(P＝0.049)。痰标本检测Rb2／p130基因突变作为肺癌诊断分子标记，特异度为100％，灵敏度为22.86％。
2 组织标本p53突变检出率43.23％(23／52)，其中NSCLC 40.91％(18／44)，SCLC 62.50％(5／8)，两者差异无显著意义(P＝0.067)。Exon5～6检出10例次、Exon7突变6例次、Exon8～9突变9例次，一例同时并Exon5～6和Exon8～9突变，另一例同时并Exon7和Exon8～9突变。44例NSCLC p53突变检出率与患者的性别、吸烟、病理类型、疾病分期均无相关性(P＞0.05)，但在高龄(≥60岁)患者检出率高于低龄(＜60岁)患者(P＝0.01)。痰标本肺癌组p53突变检出率28.57％(10／35)，对照组未检出一例，差异有显著
Objectives
1. To probe Rb2/p130 and p53 gene mutations at their hot-spots by DHPLC analysis in Chinese lung cancer patients.
2. To evaluate the feasibility of the two gene markers as a clinical diagnostic avenue of lung cancer.
Methods
1. Genomic DNAs were extracted from 52 biopsy samples (44 of NSCLC, 8 of SCLC), 47 sputum samples (35 of lung cancer, 12 of benign lung disease), and their parallel peripheral blood lymphoid cells.
2. To subject the genomic DNAs to PCR amplification of Rb2/p130 gene at exon 19-22 and p53 gene at exon 5-9.
3. The mutations of Rb2/p130 and p53 were detected by DHPLC analyzing the PCR products.
Results
1. 52 biopsy samples with a total detection rate of 23.08%( 12/52), in which 32.00%(8/25) of adenocarcinoma, 15.79%(3/19) of squamous carcinoma, and 12.50%(l/8) of small cell carcinoma, for Rb2/p130 gene mutations were confirmed. Ten cases were detected mutations at exon 21, one at exon 20, one at exon 22, but none at exon 19. No relations were found between the total detection rate of Rb2/p130 gene mutation and the patients' sexes, ages, stages, smoking, and pathologies in 44 NSCLC (all P>0.05). Of 47 sputum samples, the Rb2/p130 gene mutation detection rates were 22.86%(8/35) in lung cancer group and 0%(0/12) in control group (P=0.049). The sensitivity and specificity were 22.86% and 100% respectively by using Rb2/p130 gene mutation detected in sputum sample as a diagnostic marker for lung cancer.
1 DHPLC分析肺癌患者组织标本Rb2／p130和p53突变。
2 DHPLC分析肺癌患者痰标本Rb2／p130和p53突变及其作为临床分子诊断标记的可行性。
[方法]
1 基因组DNA提取自52例肺癌组织标本(44例NSCLC，8例SCLC)和47例痰标本(35例肺癌，12例对照)以及相应的外周血淋巴细胞。
2 PCR扩增Rb2／p130基因Exon19～22和p53基因Exon5～9。
3 DHPLC分析PCR扩增产物检测Rb2／p130和p53基因突变。
[结果]
1 组织标本Rb2／p130突变检出率23.08％(12／52)，其中腺癌32.00％(8／25)、鳞癌15.79％(3／19)、小细胞癌12.50％(1／8)，三者差异无显著意义(P＝0.403)。Exon21突变10例次，Exon20和Exon22突变各1例，未见Exon19突变。44例NSCLC Rb2／p130突变检出率与患者的性别、年龄、吸烟、病理类型、疾病分期等均无相关性(所有P＞0.05)。痰标本肺癌组Rb2／p130突变检出率22.86％(8／35)，对照组未检出一例，差异有显著意义(P＝0.049)。痰标本检测Rb2／p130基因突变作为肺癌诊断分子标记，特异度为100％，灵敏度为22.86％。
2 组织标本p53突变检出率43.23％(23／52)，其中NSCLC 40.91％(18／44)，SCLC 62.50％(5／8)，两者差异无显著意义(P＝0.067)。Exon5～6检出10例次、Exon7突变6例次、Exon8～9突变9例次，一例同时并Exon5～6和Exon8～9突变，另一例同时并Exon7和Exon8～9突变。44例NSCLC p53突变检出率与患者的性别、吸烟、病理类型、疾病分期均无相关性(P＞0.05)，但在高龄(≥60岁)患者检出率高于低龄(＜60岁)患者(P＝0.01)。痰标本肺癌组p53突变检出率28.57％(10／35)，对照组未检出一例，差异有显著
Objectives
1. To probe Rb2/p130 and p53 gene mutations at their hot-spots by DHPLC analysis in Chinese lung cancer patients.
2. To evaluate the feasibility of the two gene markers as a clinical diagnostic avenue of lung cancer.
Methods
1. Genomic DNAs were extracted from 52 biopsy samples (44 of NSCLC, 8 of SCLC), 47 sputum samples (35 of lung cancer, 12 of benign lung disease), and their parallel peripheral blood lymphoid cells.
2. To subject the genomic DNAs to PCR amplification of Rb2/p130 gene at exon 19-22 and p53 gene at exon 5-9.
3. The mutations of Rb2/p130 and p53 were detected by DHPLC analyzing the PCR products.
Results
1. 52 biopsy samples with a total detection rate of 23.08%( 12/52), in which 32.00%(8/25) of adenocarcinoma, 15.79%(3/19) of squamous carcinoma, and 12.50%(l/8) of small cell carcinoma, for Rb2/p130 gene mutations were confirmed. Ten cases were detected mutations at exon 21, one at exon 20, one at exon 22, but none at exon 19. No relations were found between the total detection rate of Rb2/p130 gene mutation and the patients' sexes, ages, stages, smoking, and pathologies in 44 NSCLC (all P>0.05). Of 47 sputum samples, the Rb2/p130 gene mutation detection rates were 22.86%(8/35) in lung cancer group and 0%(0/12) in control group (P=0.049). The sensitivity and specificity were 22.86% and 100% respectively by using Rb2/p130 gene mutation detected in sputum sample as a diagnostic marker for lung cancer.
引文
1 李龙芸 继续深入开展肺癌的基础与临床研究 中华内科杂志2000；39 (11)：725-727。
2 Wingo PA, Ries LA, Giovino GA, et al. Annual report to the nation on the status of cancer, 1973-1996,with a special section on lung cancer and tobacco smoking. JNatl Cancerlnst 1999; 91: 675-690.
3 Travis WD, Travis LB, Devesa SS. Lung cancer. Cancer 1995; 75: 191-202.
4 Bailar JC. Early lung cancer cooperative study group: Early lung cancer detection. Summary and conclusion. Am Rev Respir DIS 1984; 130:565-571.
5 Lang SM, Stratakis DF, Freudling A, et al: Detection of K-ras and p53 mutation in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Eur J Med Res 2000; 5(8): 341-346.
6 Claudio PP, De Luca A, Howard CM, et al: Functional analysis ofpRb2/p130 interaction with cyclins. Cancer Res 1996; 56: 2003-2008.
7 Paggi MG, Baldi A, Bonetto F, et al. Retinoblastoma protein family in cell cycle and cancer: a review. J Cellular Biochemistry 1996; 62: 418-430.
8 Claudio PP, Howard CM, Baldi A, et al. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res 1994; 54: 5556-5560.
9 Yeung RS, Bell DW, Testa JR, et al.: The retinoblastoma-related??gene,RB2,maps to human chromosome 16q12 and rat chromosome 19. Oncogene 1993; 8: 3465-3468.
10 Baldi A, Esposito V, De Luca A, et al: Differential expression of the retinoblastoma gene family members, Rb/p105, p107 and Rb2/p130, in lung cancer. Clin Cancer Res 1996; 7: 1239-1245.
11 Claudio PP, Howard CM, Pacilio C, et al: Mutations in the retinoblastoma-related gene Rb2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 2000; 60: 372-382.
12 Helin K, Holm K, Niebuhr A, et al: Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma. Proc NatlAcad Sci USA 1997; 94: 6933-6938.
13 Yandell DW, Campbell TA, Dayton SH, et al: Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling, N Engl J Med 1989; 321: 1689-1695.
14 Baldi A, Boccia V, Claudio PP, et al: Genomic structure of the human retinoblastoma-related Rb2/pl30 gene. Proc Natl Acad Sci USA 1996; 93: 4629-4632.
15 Oefner P, Underhill P. Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am J Hum Genet 1995; 57(suppl):A266.
16 Conti CJ. Application of high-performance liquid chromatography-based analysis of DNA fragments to molecular carcinogenesis. Molecular??Carcinogenesis 2000; 29:51-58.
17 O'Donovan MC, Oefner PJ, Roberts SC, et al. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998; 52: 44-49.
18 Waga S, Harmon GJ, Beach D, and Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 1994, 369: 574-578.
19 Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell 1998; 95: 1437-1443.
20 Smith ML,. Chen IT, Zhan Q, et al. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 1994; 266: 1376-1380.
21 Cho Y, Gorina S, Jeffery P, et al. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 1994; 265: 346-355.
22 Levine AJ, Momand J, and Finlay CA. The p53 tumor suppressor gene. Nature 1991; 351: 453-456.
23 Ramet M, Casten K, Jarvinen K et al. P53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines. Carcinogenesis 1996; 16: 2116-2124.
24 Denissenko MF, Pao A, Tang M, and Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 1996; 274: 430-432.25 Chen JX, Zheng Y, West M, andTang M. Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res 1998; 58: 2070-2075.
26 Lang SM, Stratakis DF, Freuding A, et al. Detection of K-ras and p53 mutations in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Fur J Med Res 2000; 5(8): 341-346.
27 Li Mao, Hruban AH, Boyle JO, et al. Detection of oncongene mutation in sputum precedes diagnosis of lung cancer. Cancer Res 1994; 54: 1634-1637.
28 汪斌超，李龙芸，姚连昌等。痰标本检测p53基因突变及其在肺癌早期临 床诊断中的意义。中华内科志，2001：40(2)：101-104。
29 Bennett WP, Colby TV, Travis WD, et al. p53 protein accumulates frequently in the early bronchial neoplasia. Cancer Res 1993; 53: 4817-4822.
30 Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996; 56: 2224-2228.
31 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16INK4A is an early event in lung cancer and a potential biomarker for early diagnosis. ProcNatlAcadSci USA 1998; 95: 11891-11896.
32 李龙芸，汪斌超，陈勇等改良Saccomanno法用于肺癌患者痰标本处理的 应用体会 中国肺癌杂志2000；3(4)：280—2831 Landis SH, Murray T, Bolden S, et al: Cancer Stastistics. CA Cancer J Clin 1999; 49: 8-31.
2 Franceschi S, Bidoli E. The epidemiology of lung cancer. Annals of Oncology 1999; 10 (Suppl.5): s3-s6.
3 Wuerin J and P Nurse. Regulating S phase: CDKs, licensing and proteolysis. Cell 1996; 85: 185-787.
4 Sherr C. Cancer cell cycles. Science 1996; 274: 1672-1677.
5 Hunter T. and J Pines. Cyclins and cancer IIL cyclin D and CDK inhibitors come of age. Cell 1994; 79:573-582.
6 Weintraub S.J. Inactivation of tumor suppressor proteins in lung cancer. Am. J. Respir. Cell Mol. Biol. 1996; 15: 150-155.
7 E1-Deiry, W. S., T Tokino, V. E. Velculescu, et al. WAF 1. A potential mediator of p53 tumor suppression. Cell 1993; 75: 817-825.
8 Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 1992;258: 424-429.
9 Stillman B. Cell cycle control of DNA replication. Science 1996; 274: 1659-1663.
10 Claudio PP, Howard CM, Pacilio C, et al. Mutations in the retinoblastoma-related gene RB2/pl30 in lung tumors and suppression of??tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 2000; 60: 372-382.
11 Horowitz JM. Regulation of transcription by the retinoblastoma protein. Genes Chromosom. Cancer 1993; 6: 124-131.
12 Ludlow JW, Glendening CL, Livingston DM, et al. Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Bial 1993; 13: 367-372.
13 Hinds PW, Mittnacht S, Dulic V, et al. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 1992; 70: 993-1006.
14 Harper JW, Adami GR, Wei N et al. The p21 Cdk-interacting protein Cipl is a potent inhibitor of Gl cyclin-dependent kinases. Cell 1993; 75: 805-
15 Paggi MG, Baldi A, Bonetto F, et al. Retinoblastoma protein family in cycle and cancer: a review. J Cellular Bioche 1996; 62: 418-430.
16 Kwun M, Paul V, Peter J. Lung pathology: the molecular genetics of non-small cell lung cancer. Pathology, 1995; 27: 295-301.
17 Yeung Rs, Bell DW, Testa JR, et al. The retinoblastoma-related gene, Rb2, maps to human chromosome 16q12 and rat chromosome 19. Oncogene 1993; 8: 3465-3468.
18 Biggs PJ, Wooster R, Ford D, et al. Familial cyclindromatosis (turban tumor syndrome) gene localised to chromosome 16q12-q13: evidence for its role as a tumor suppressor gene. Nat Genet 1995; 11: 441-443.19 Levine AJ, Momand J, and Finlay CA. The p53 tumor suppressor gene. Nature 1991; 351: 453-456.
20 Ramet M, Casten K, Jarvinen K et al. P53 protein expression is correlated with benzo [a] pyrene-DNA adduets in carcinoma cell lines. Carcinogenesis 1996; 16: 2116-2124.
21 Denissenko M F, Pao A, "Tang M, and Pfeifer GP. Preferential formation of benzo [a] pyrene adducts at lung cancer mutational hotspots in p53. Science 1996; 274: 430-432.
22 Chen JX, Zheng Y, West M, andTang M. Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res 1998; 58: 2070-2075.
23 Waga S, Hannon GJ, Beach D, and Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 1994, 369: 574-578.
24 Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell 1998; 95: 1437-1443.
25 Barak Y, Juven T, Haffner R and Oren M. Mdm2 expression is induced by wild type p53 activity. EMBO J. 1993; 12: 461-468.
26 Smith ML,. Chen IT, Zhan Q, et al. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 1994; 266: 1376-1380.
27 Zambetti G, and Levine AJ. A comparison of the biological activities of wild-type and mutant p53. FASEB J. 1993; 7: 855-865.28 Lang SM, Stratakis DF, Freuding A, et al. Detection of K-ras and p53 mutations in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Eur J Med Res 2000; 5(8): 341-346.
29 Kamb A, Gruits NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994; 264: 436-440.
30 Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell cycle control causing specific inhibition ofcyclin D/CDK4. Nature. 1994; 366(6456): 704-
31 Otterson GA, Kratzke RA, Coxon A, et al. Absence of p16 protein is restricted to the subset of lung cancer lines that retains wildtype RB. Oncogene. 1994; 9(16): 3375-
32 Shapiro GI, Edwards CD, Kobzik L, et al. Reciprocal Rb inactivation and p16~(INK4) expression in primary lung cancers and cell lines. Cancer Res 1995; 55: 505-509.
33 Okamoto A, Hussain SP, Hagiwara K, et al. Mutations in the p16INK4/MTS1/CDKN2, p16INK4B/MTS2, and p18 genes in primary and metastatic lung cancer. Cancer Res 1995; 55: 1448-1451.
34 Rusin M, Okamoto RA, Cjorazy M, et al. Intragenic mutations of the p16~(INK4), p15~(INK4B) and p18 genes in primary non-small-cell lung casneers. Int. J. Cancer 1996; 65: 734-739.
35 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of??p16~(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11891-11896.
36 Haber DA. Splicing into senescence: the curious case of p16 and p19~(ARF). Cell 1997; 91: 555-558.
37 Bates S, Phillips A, Clarke P, et al. p14~(ARF) links the tumour suppressors Rb and p53. Nature 1998; 395: 125-125.
38 Pomerantz J, Schreiber-Angus N, Liegeois NJ, et al. The INK4a tumor suppressor gene product, p19~(ARF), interacts with MCM2 and neutralizes MDM2's inhibition of p53. Cell 1998; 92: 713-723.
39 Kamijo T. Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Nat. Acad. Sci. U. S. A. 1998;. 95: 8292-8297.
40 Zindy F, Eischen CM, Handle DH et al. Myc signaling via the ARF rumor suppressor rugulates p53-dependent apoptosis and immortalization. Genes Devel. 1998; 12: 2424-2433.
41 Gazzeri S, Della Valle V, Chaussade L, et al. The human p19~(INK4a) gene is frequently lost in small cell lung cancer. Cancer Res 1998; 58: 3926-3931.
42 Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t (3; 8) breakpoint, is abnormal in digestive tract cancers. Cell 1996; 84(4): 587-597.
43 Barnes LD, Garrison PN, Siprashvili Z, et al. Fhit, a putative tumor suppressor in humans, is a dinucleoside 5', 5'"-p1, p3-triphosphate hydrolase. Biochemistry??1996; 35(36): 11529-11535.
44 Sard L, Accornero P, Torniellie S, et al. The tumor suppressor gene FHIT is inxolved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci USA. 1999; 96(15): 8489-8492.
45 Ji L, Fang B, Yen N, et al. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res. 1999; 59(14): 3333-3339.
46 Shi Y, Zou M, Farid NR, et al. Association of FHIT, a candidate tumor suppressor gene, with the ubiquitin-conjugating enzyme hUBC9. Biochemistry 2000; 352pt2: 443-448.
47 Tanaka H, Shimada Y, Harada H, et al. Methylation of the 5' CpG island of the FHIT gene is closely associated with transcriptional inactivation in esophageal squamous cell carcinomas. Cancer Res. 1998; 58(15): 3429-3434.
48 Sozzi G, Veronese ML, Negrini M, et al. The Fhit gene 3p14.. 2 is abnormal in lung cancer. Cell 1996; 85(1): 17-26.
49 Sozzi G, Pastorino U, Moiraghi L, et al. Loss of Fhit function in lung cancer and preinvasive bronchial lesions. Cancer Res 1998; 58(22): 5032-5037.
50 Heldin CH, Kohei M, and Peter D. TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature 1997; 390: 465-471.
51 Harmon GJ, and Beach K. P15~(INK4B) is a potential effector of TGF-β-induced cell cycle arrest. Nature 1994; 371: 257-261.52 Kin SJ, Wagner S, Lin F, et al. Retinoblastoma gene product activates expression of the human TGF-β_2 gene through transcription factor ATF-2. Nature 1992; 358: 331-334.
53 Iavarone A and Massague J. Repression of the CDK activator Cdc 25A and cell-cycle arrest by cytokine TGF-β in cells lacking the CDK inhibitor p15. Nature 1997; 387: 416-421.
54 Peeper DS, Upton TM, Ladha M, et al. Ras signaling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 1997; 386: 177-181.
55 Sears R, Leone G, DeGregori J, et al. Ras enhances myc protein stability. Mol. Cell 1999; 3: 169-179.
56 Leone G, DeGregori J, Sears R, et al. Myc and ms collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 1997; 387: 422-426.
57 Serrano M, Gomez-: ahoz E, Depinbo RA, et al. Inhibition of ras-induced proliferation and cellular transformation by p16~(ink4). Science 1995; 267: 249-252.
58 Li. ZH, Zheng J, Weiss LM, et al. K-ras and p53 mutations occur very early in adenocarcinoma of the lung. Am. J. Pathol. 1994; 144: 303-309.
59 Rodenhuis S, Slebos RJ. Incidence and possible clinical significance of ras activation in adenocacinoma of the human lung. Cancer Res I988; 48: 5738-5741.
60 Galaktionov K, Chen X, and Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 1996; 382: 511-517.61 Evan GI, Wyllie AH, Gilbert CS et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992; 69: 119-128.
62 Wagner AJ,. Kokontis JM, Hay N. Myc-mediated apoptosis requires wild-typep53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Gene & development 1994; 8: 2817-2830.
63 Lee YC, Li L, philipson L, et al. Myc represses transcription of the growth arrest gene gasdl. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12886-12891.
64 Del Sal G, Ruaro EM, Utrem R, et al. Gasl-induced growth suppression requires a transactivation-independent p53 function. Mol. Ceil Biol. 1995; 15: 7152-7160.
65 Viallet J, and Minna J. Dominant oncogenes and tumor suppressor genes in the pathogenesis of human lung cancer. Am. J. Respir. Cell Mol. Bil. 1990; 2: 225-232.
66 Arzimanoglou H, Gilbert F, Barber. Microsatellite instability in human solid tumor. Cancer 1998; 82: 1808-1820.
67 Mao Li, Sehoenberg MP, Seicechitano M, et al. Molecular detection of primary bladder cancer by Mierosatellite analysis. Science 1996; 271: 659-662.
68 Steiner G, Schoenberg MP, Linn JF, et al. Detection of bladder cancer recurrence by microsatellite analysis of urine. J Nat Med 1997; 3: 621-624.
69 Sauchez-Cespedes M, Monzo M, Rosell R, et al. Detection of chromosome 3p alteration in serum DNA of non-small-cell lung cancer patients. J Ann Oncol 1998; 9: 113-116.
2 Wingo PA, Ries LA, Giovino GA, et al. Annual report to the nation on the status of cancer, 1973-1996,with a special section on lung cancer and tobacco smoking. JNatl Cancerlnst 1999; 91: 675-690.
3 Travis WD, Travis LB, Devesa SS. Lung cancer. Cancer 1995; 75: 191-202.
4 Bailar JC. Early lung cancer cooperative study group: Early lung cancer detection. Summary and conclusion. Am Rev Respir DIS 1984; 130:565-571.
5 Lang SM, Stratakis DF, Freudling A, et al: Detection of K-ras and p53 mutation in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Eur J Med Res 2000; 5(8): 341-346.
6 Claudio PP, De Luca A, Howard CM, et al: Functional analysis ofpRb2/p130 interaction with cyclins. Cancer Res 1996; 56: 2003-2008.
7 Paggi MG, Baldi A, Bonetto F, et al. Retinoblastoma protein family in cell cycle and cancer: a review. J Cellular Biochemistry 1996; 62: 418-430.
8 Claudio PP, Howard CM, Baldi A, et al. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res 1994; 54: 5556-5560.
9 Yeung RS, Bell DW, Testa JR, et al.: The retinoblastoma-related??gene,RB2,maps to human chromosome 16q12 and rat chromosome 19. Oncogene 1993; 8: 3465-3468.
10 Baldi A, Esposito V, De Luca A, et al: Differential expression of the retinoblastoma gene family members, Rb/p105, p107 and Rb2/p130, in lung cancer. Clin Cancer Res 1996; 7: 1239-1245.
11 Claudio PP, Howard CM, Pacilio C, et al: Mutations in the retinoblastoma-related gene Rb2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 2000; 60: 372-382.
12 Helin K, Holm K, Niebuhr A, et al: Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma. Proc NatlAcad Sci USA 1997; 94: 6933-6938.
13 Yandell DW, Campbell TA, Dayton SH, et al: Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling, N Engl J Med 1989; 321: 1689-1695.
14 Baldi A, Boccia V, Claudio PP, et al: Genomic structure of the human retinoblastoma-related Rb2/pl30 gene. Proc Natl Acad Sci USA 1996; 93: 4629-4632.
15 Oefner P, Underhill P. Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am J Hum Genet 1995; 57(suppl):A266.
16 Conti CJ. Application of high-performance liquid chromatography-based analysis of DNA fragments to molecular carcinogenesis. Molecular??Carcinogenesis 2000; 29:51-58.
17 O'Donovan MC, Oefner PJ, Roberts SC, et al. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998; 52: 44-49.
18 Waga S, Harmon GJ, Beach D, and Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 1994, 369: 574-578.
19 Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell 1998; 95: 1437-1443.
20 Smith ML,. Chen IT, Zhan Q, et al. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 1994; 266: 1376-1380.
21 Cho Y, Gorina S, Jeffery P, et al. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 1994; 265: 346-355.
22 Levine AJ, Momand J, and Finlay CA. The p53 tumor suppressor gene. Nature 1991; 351: 453-456.
23 Ramet M, Casten K, Jarvinen K et al. P53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines. Carcinogenesis 1996; 16: 2116-2124.
24 Denissenko MF, Pao A, Tang M, and Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 1996; 274: 430-432.25 Chen JX, Zheng Y, West M, andTang M. Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res 1998; 58: 2070-2075.
26 Lang SM, Stratakis DF, Freuding A, et al. Detection of K-ras and p53 mutations in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Fur J Med Res 2000; 5(8): 341-346.
27 Li Mao, Hruban AH, Boyle JO, et al. Detection of oncongene mutation in sputum precedes diagnosis of lung cancer. Cancer Res 1994; 54: 1634-1637.
28 汪斌超，李龙芸，姚连昌等。痰标本检测p53基因突变及其在肺癌早期临 床诊断中的意义。中华内科志，2001：40(2)：101-104。
29 Bennett WP, Colby TV, Travis WD, et al. p53 protein accumulates frequently in the early bronchial neoplasia. Cancer Res 1993; 53: 4817-4822.
30 Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996; 56: 2224-2228.
31 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16INK4A is an early event in lung cancer and a potential biomarker for early diagnosis. ProcNatlAcadSci USA 1998; 95: 11891-11896.
32 李龙芸，汪斌超，陈勇等改良Saccomanno法用于肺癌患者痰标本处理的 应用体会 中国肺癌杂志2000；3(4)：280—2831 Landis SH, Murray T, Bolden S, et al: Cancer Stastistics. CA Cancer J Clin 1999; 49: 8-31.
2 Franceschi S, Bidoli E. The epidemiology of lung cancer. Annals of Oncology 1999; 10 (Suppl.5): s3-s6.
3 Wuerin J and P Nurse. Regulating S phase: CDKs, licensing and proteolysis. Cell 1996; 85: 185-787.
4 Sherr C. Cancer cell cycles. Science 1996; 274: 1672-1677.
5 Hunter T. and J Pines. Cyclins and cancer IIL cyclin D and CDK inhibitors come of age. Cell 1994; 79:573-582.
6 Weintraub S.J. Inactivation of tumor suppressor proteins in lung cancer. Am. J. Respir. Cell Mol. Biol. 1996; 15: 150-155.
7 E1-Deiry, W. S., T Tokino, V. E. Velculescu, et al. WAF 1. A potential mediator of p53 tumor suppression. Cell 1993; 75: 817-825.
8 Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 1992;258: 424-429.
9 Stillman B. Cell cycle control of DNA replication. Science 1996; 274: 1659-1663.
10 Claudio PP, Howard CM, Pacilio C, et al. Mutations in the retinoblastoma-related gene RB2/pl30 in lung tumors and suppression of??tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res 2000; 60: 372-382.
11 Horowitz JM. Regulation of transcription by the retinoblastoma protein. Genes Chromosom. Cancer 1993; 6: 124-131.
12 Ludlow JW, Glendening CL, Livingston DM, et al. Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Bial 1993; 13: 367-372.
13 Hinds PW, Mittnacht S, Dulic V, et al. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 1992; 70: 993-1006.
14 Harper JW, Adami GR, Wei N et al. The p21 Cdk-interacting protein Cipl is a potent inhibitor of Gl cyclin-dependent kinases. Cell 1993; 75: 805-
15 Paggi MG, Baldi A, Bonetto F, et al. Retinoblastoma protein family in cycle and cancer: a review. J Cellular Bioche 1996; 62: 418-430.
16 Kwun M, Paul V, Peter J. Lung pathology: the molecular genetics of non-small cell lung cancer. Pathology, 1995; 27: 295-301.
17 Yeung Rs, Bell DW, Testa JR, et al. The retinoblastoma-related gene, Rb2, maps to human chromosome 16q12 and rat chromosome 19. Oncogene 1993; 8: 3465-3468.
18 Biggs PJ, Wooster R, Ford D, et al. Familial cyclindromatosis (turban tumor syndrome) gene localised to chromosome 16q12-q13: evidence for its role as a tumor suppressor gene. Nat Genet 1995; 11: 441-443.19 Levine AJ, Momand J, and Finlay CA. The p53 tumor suppressor gene. Nature 1991; 351: 453-456.
20 Ramet M, Casten K, Jarvinen K et al. P53 protein expression is correlated with benzo [a] pyrene-DNA adduets in carcinoma cell lines. Carcinogenesis 1996; 16: 2116-2124.
21 Denissenko M F, Pao A, "Tang M, and Pfeifer GP. Preferential formation of benzo [a] pyrene adducts at lung cancer mutational hotspots in p53. Science 1996; 274: 430-432.
22 Chen JX, Zheng Y, West M, andTang M. Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res 1998; 58: 2070-2075.
23 Waga S, Hannon GJ, Beach D, and Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 1994, 369: 574-578.
24 Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell 1998; 95: 1437-1443.
25 Barak Y, Juven T, Haffner R and Oren M. Mdm2 expression is induced by wild type p53 activity. EMBO J. 1993; 12: 461-468.
26 Smith ML,. Chen IT, Zhan Q, et al. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 1994; 266: 1376-1380.
27 Zambetti G, and Levine AJ. A comparison of the biological activities of wild-type and mutant p53. FASEB J. 1993; 7: 855-865.28 Lang SM, Stratakis DF, Freuding A, et al. Detection of K-ras and p53 mutations in bronchoscopically obtained malignant and non-malignant tissue from patients with non-small cell lung cancer. Eur J Med Res 2000; 5(8): 341-346.
29 Kamb A, Gruits NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994; 264: 436-440.
30 Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell cycle control causing specific inhibition ofcyclin D/CDK4. Nature. 1994; 366(6456): 704-
31 Otterson GA, Kratzke RA, Coxon A, et al. Absence of p16 protein is restricted to the subset of lung cancer lines that retains wildtype RB. Oncogene. 1994; 9(16): 3375-
32 Shapiro GI, Edwards CD, Kobzik L, et al. Reciprocal Rb inactivation and p16~(INK4) expression in primary lung cancers and cell lines. Cancer Res 1995; 55: 505-509.
33 Okamoto A, Hussain SP, Hagiwara K, et al. Mutations in the p16INK4/MTS1/CDKN2, p16INK4B/MTS2, and p18 genes in primary and metastatic lung cancer. Cancer Res 1995; 55: 1448-1451.
34 Rusin M, Okamoto RA, Cjorazy M, et al. Intragenic mutations of the p16~(INK4), p15~(INK4B) and p18 genes in primary non-small-cell lung casneers. Int. J. Cancer 1996; 65: 734-739.
35 Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of??p16~(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11891-11896.
36 Haber DA. Splicing into senescence: the curious case of p16 and p19~(ARF). Cell 1997; 91: 555-558.
37 Bates S, Phillips A, Clarke P, et al. p14~(ARF) links the tumour suppressors Rb and p53. Nature 1998; 395: 125-125.
38 Pomerantz J, Schreiber-Angus N, Liegeois NJ, et al. The INK4a tumor suppressor gene product, p19~(ARF), interacts with MCM2 and neutralizes MDM2's inhibition of p53. Cell 1998; 92: 713-723.
39 Kamijo T. Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Nat. Acad. Sci. U. S. A. 1998;. 95: 8292-8297.
40 Zindy F, Eischen CM, Handle DH et al. Myc signaling via the ARF rumor suppressor rugulates p53-dependent apoptosis and immortalization. Genes Devel. 1998; 12: 2424-2433.
41 Gazzeri S, Della Valle V, Chaussade L, et al. The human p19~(INK4a) gene is frequently lost in small cell lung cancer. Cancer Res 1998; 58: 3926-3931.
42 Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t (3; 8) breakpoint, is abnormal in digestive tract cancers. Cell 1996; 84(4): 587-597.
43 Barnes LD, Garrison PN, Siprashvili Z, et al. Fhit, a putative tumor suppressor in humans, is a dinucleoside 5', 5'"-p1, p3-triphosphate hydrolase. Biochemistry??1996; 35(36): 11529-11535.
44 Sard L, Accornero P, Torniellie S, et al. The tumor suppressor gene FHIT is inxolved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci USA. 1999; 96(15): 8489-8492.
45 Ji L, Fang B, Yen N, et al. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res. 1999; 59(14): 3333-3339.
46 Shi Y, Zou M, Farid NR, et al. Association of FHIT, a candidate tumor suppressor gene, with the ubiquitin-conjugating enzyme hUBC9. Biochemistry 2000; 352pt2: 443-448.
47 Tanaka H, Shimada Y, Harada H, et al. Methylation of the 5' CpG island of the FHIT gene is closely associated with transcriptional inactivation in esophageal squamous cell carcinomas. Cancer Res. 1998; 58(15): 3429-3434.
48 Sozzi G, Veronese ML, Negrini M, et al. The Fhit gene 3p14.. 2 is abnormal in lung cancer. Cell 1996; 85(1): 17-26.
49 Sozzi G, Pastorino U, Moiraghi L, et al. Loss of Fhit function in lung cancer and preinvasive bronchial lesions. Cancer Res 1998; 58(22): 5032-5037.
50 Heldin CH, Kohei M, and Peter D. TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature 1997; 390: 465-471.
51 Harmon GJ, and Beach K. P15~(INK4B) is a potential effector of TGF-β-induced cell cycle arrest. Nature 1994; 371: 257-261.52 Kin SJ, Wagner S, Lin F, et al. Retinoblastoma gene product activates expression of the human TGF-β_2 gene through transcription factor ATF-2. Nature 1992; 358: 331-334.
53 Iavarone A and Massague J. Repression of the CDK activator Cdc 25A and cell-cycle arrest by cytokine TGF-β in cells lacking the CDK inhibitor p15. Nature 1997; 387: 416-421.
54 Peeper DS, Upton TM, Ladha M, et al. Ras signaling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 1997; 386: 177-181.
55 Sears R, Leone G, DeGregori J, et al. Ras enhances myc protein stability. Mol. Cell 1999; 3: 169-179.
56 Leone G, DeGregori J, Sears R, et al. Myc and ms collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 1997; 387: 422-426.
57 Serrano M, Gomez-: ahoz E, Depinbo RA, et al. Inhibition of ras-induced proliferation and cellular transformation by p16~(ink4). Science 1995; 267: 249-252.
58 Li. ZH, Zheng J, Weiss LM, et al. K-ras and p53 mutations occur very early in adenocarcinoma of the lung. Am. J. Pathol. 1994; 144: 303-309.
59 Rodenhuis S, Slebos RJ. Incidence and possible clinical significance of ras activation in adenocacinoma of the human lung. Cancer Res I988; 48: 5738-5741.
60 Galaktionov K, Chen X, and Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 1996; 382: 511-517.61 Evan GI, Wyllie AH, Gilbert CS et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992; 69: 119-128.
62 Wagner AJ,. Kokontis JM, Hay N. Myc-mediated apoptosis requires wild-typep53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Gene & development 1994; 8: 2817-2830.
63 Lee YC, Li L, philipson L, et al. Myc represses transcription of the growth arrest gene gasdl. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12886-12891.
64 Del Sal G, Ruaro EM, Utrem R, et al. Gasl-induced growth suppression requires a transactivation-independent p53 function. Mol. Ceil Biol. 1995; 15: 7152-7160.
65 Viallet J, and Minna J. Dominant oncogenes and tumor suppressor genes in the pathogenesis of human lung cancer. Am. J. Respir. Cell Mol. Bil. 1990; 2: 225-232.
66 Arzimanoglou H, Gilbert F, Barber. Microsatellite instability in human solid tumor. Cancer 1998; 82: 1808-1820.
67 Mao Li, Sehoenberg MP, Seicechitano M, et al. Molecular detection of primary bladder cancer by Mierosatellite analysis. Science 1996; 271: 659-662.
68 Steiner G, Schoenberg MP, Linn JF, et al. Detection of bladder cancer recurrence by microsatellite analysis of urine. J Nat Med 1997; 3: 621-624.
69 Sauchez-Cespedes M, Monzo M, Rosell R, et al. Detection of chromosome 3p alteration in serum DNA of non-small-cell lung cancer patients. J Ann Oncol 1998; 9: 113-116.