人结肠癌细胞系对5-氟尿嘧啶（5-FU）与顺铂（CDDP）敏感性相关分子标记研究
摘要
在过去二十年里，在分子水平解释癌发生机制取得了巨大的进步，但仍然存在不少问题，随着人类寿命的延长，在一些国家问题变得更加严峻。在结直肠癌研究方面，根据不同的分子生物学和临床特征，结直肠癌大概可以被分为三类：家族性腺瘤性息肉病相关结直肠癌(Familial Adenomatous Polyposis-associated Colorectal Cancer，FAP-CRC)，遗传性非息肉性结直肠癌(Hereditary Non-Polyposis ColonCancer，HNPCC)，散发性结直肠癌(Sporadic Colorectal Cancer，SCRC)，已有的研究资料表明，它们具有不同的分子特征。
对结直肠癌的治疗方面，目前仍然采用手术、化疗和放疗相结合的综合治疗手段，所用的化疗方案主要是以5-氟尿嘧啶(5-FU)为基础的多个药物组合，临床上实践证明化疗能杀灭术前或术后发生的微转移灶，从而有效地降低进展期病人死亡率，但仍然离我们的期望甚远，目前报道的以5-FU为基础的各种化疗方案的有效率基本上在15～21％之间，可能与低有效率相关的生化机制主要涉及六大类化疗敏感性相关因子，包括药物外排，灭活、损伤DNA的修复，药物作用靶点的调节和其它(如凋亡，端粒长度和端粒酶活性等)。此外，临床回顾性研究还发现大约半数Dukes’C期的CRC病人并不真正需要化疗，而约35％的化疗病人并没有真正得益于系统性化疗。由此表明，对化疗预后或疗效预测分子标记的研究深具意义，它不仅可以提供CRC化疗病人预后信息，还为特定病例化疗与否，化疗方案的选择，即“个体化化疗”提供依据，随着生物芯片技术的发展，这方面的工作愈加显得重要。
顺铂(CDDP)是一种铂类衍生物，它在肺癌、卵巢癌等化疗中被广泛使用。目前在一些医院的肿瘤科与5-FU一起用于CRC病人化疗。由于这两种药物作用机制完全不同，因此被认为能够在联合用药中起到协同作用而提高疗效。但是，
CDDP临床耐药报道较多．临床疗效差异牲极大．因此推测可能存在明确的化疗
敏感相矢机制。
为了寻找与结肠扬细胞系5千U和／或CDDP化疗敏脓牲相矢因子，探讨5－FU
和／或CDDP化疗敏成牲生化机制．我们在此研究了三个人结肠癌细胞系对5－FU
和／或CDDP反应表型、在5－FU和／或CDDP作用下细胞局期分布、胞苦酸台成酶
（TS）mRNA水平、P53、错配修复蛋白hMSHZ、hMLHI表达状况．以及十个与细
胞增殖或死亡相矢的微卫皇位点状况。通过比较药物欧感牲表型和上述遗传背景
之间的夫系，以揭示或部分阐明CRC病人预后或预测分子标记在用药优化和预后
评价中的作用。
第一韶分
三个人结＆＄细胞系对SFU和l或 CDDP处理的敏感性表型及 P53蛋白表达
在这个部分我们首先通过形态学观寨、MTT ＃活牲测定和硫式细胞分析仪检
测了结肠癌细胞系LOVO、SW480和SWill6细胞对5下U和／或CDDP处理的反应．
周期阻滞倩况；并用免疫细胞化学方法检测P53蛋臼在这三个细胞系中的表达倩
况。
结果发现5干U和CDDP M三个细胞系都表现出不同程度的毒牲作用。形态学
观察表明 LOVO细胞在 8 P M或 64 P M的 5－FU作用下出现明显的毒牲反应：核固络、
变圆、折光牲增加．以及细胞数目明显减少；而SW480和SWill6细胞的毒枉反
应则相对较轻些。细胞活力分析也表明．刘低剂云（＜4厂M）处理肘．三个细胞
系中L。Vo细胞刘5·FU最敏感．SW480细胞最不敏感．SWI 116介于两者之间。在
8 P M时．也表现为 LOVo最敏感。在处理后 12小肘，LOVO细胞的 5－FU IC。。值明显
低于其它两个细胞系（分别为：0二2 p M、2．2 u M、1．9 u M，P二0刀加）。肘间－反应曲
线提示．三个细胞系对5－FU的反应有时间依赖性．但LoVO细胞仍然投另两个细
胞系敏感。研究表明SW480和LoVo细胞对CDDP的反应诌具有一定敏感牲．但前
者略比后者欧感．而SWill6则对CDDP耐受。三个细胞系对5－FU＋CDDP的联台应
用都没有表现出协同作用。
细胞周期分析表明．在5－FU作用下．LOVO细胞系出现亚二倍体峰时间比SW480
细胞系提前48小阿；而CDDP作用48小肘后，SW480细胞系比L。V。细胞系出现
更多的亚二倍体降细胞，作用96小肘后，前者亚二倍体蟹细胞数目接近
xi
后者的 2倍（分别为 55．1％和 28．5％）；当两种药物联台使用肘．亚二倍体降细胞
数目明显凉少。此外．两个纫胞系在5－FU作用下诌表现为GIM阻滞，而S M细
胞数目纫下降：但CDDP的作用却刚好相反，它主要使细胞阻滞在S期；两药联
台应用肘，细胞周期分布障药物作用肘间延长出现较大的波动。
P53免疫组织化学染色发现，L。VO细胞系细胞出现散在的巢状分布．核染色
细胞数目少于1％；而SW480和SWill6细胞细胞核出现明显的诌一染色，阳性细
胞数目超过98％。
莆二韶分
SFU和／或CDDP作用下三个结肠癌细胞系胸旮酸合成酶（TS）mRAN表达
在第一部分里，我们发现LoVO细胞系比SW480和SWill6细胞系对5－FU更加
欧感，而CDDP不尽相同。不少研究报道．肿瘤细胞对5－FU的欧感牲和脑吝酸台
成酶（TS）表达水平密切相夫。在此．我们也用 RT·PCR方法在肘间?
The last twenty years have witnessed the revolution in our JL understanding of the processes responsible for the carcinogenesis at molecular level, but the problems still exist, or even are growing as the average lifespan in many countries steadily rises. So far, colorectal cancers are usually classified into three subgroups in terms of molecular biology, genetics as well as clinical features: Familial Adenomatous Polyposis (FAP)-associated CRC, Hereditary Non-Polyposis Colon Cancer (HNPCC) and Sporadic Colorectal Cancer (SCRC), each of them has its predominant molecular portrait.
Besides surgery, radiotherapy and biological therapy, chemotherapy is proven effective in reducing mortality for advanced CRC patients by killing micrometastases disseminated at surgery or occult metastases already present at surgery. Among various regimens, combination therapies of 5-fluorouracil (5-FU) with some second-line agents have been the mainstay of adjuvant chemotherapy for CRC patients to date, but regardless of regimens, the outcome of CRC patients is far from satisfaction owing to various biochemical or non-biochemical mechanisms. In various tumors, at least five groups of sensitivity-associated factors have been identified, including drug efflux, detoxification, DMA repair, drug target regulations and other factors as apoptosis, length of telomere, etc. In addition, clinical data suggests that, in retrospect, nearly half of the patients with Dukes' C colorectal caner might not require adjuvant chemotherapy and about 35%
would die even when treated with systemic chemotherapy. This also highlights the need for informative predictive or prognostic markers that will aid identification of patients best treated with surgery alone, those requiring chemotherapy and those who might benefit from aggressive or experimental therapy.
To elucidate the possible biochemical mechanisms of chemosensitivity, and to identify possible factors involved in response to 5-FU-based regimens in CRC, we studied the chemosensitive phenotype to 5-FU, CDDP or their combination therapy, alterations of cell cycle distribution and transcription level of TS in exposure to drugs, expression of several proteins and the microsatellite status in three human colon cancer cell lines. By comparing the phenotype and their genetic background, we look forward to revealing whether these molecular events can be used as convenient predictors for drug-optimization or prognosis-assessment in CRC patients.
Part I
Phenotype of chemosensitivity to 5-fluorouracil & cisplatin and p53 expression in three human colon cancer cell lines
Firstly, we determined the phenotype of chemosensitivity to 5-fluorouracil and cisplatin in LoVo, SW480 and SW1116 cell lines in vitro, using microscopic morphology, MTT assay and Flow cytometry. Considering that big controversies exist in the biological role of p53 in chemosensitivity, we also analyzed its expression in those three cell lines by immunocytochemistry.
Results showed that 5-FU and CDDP were cytotoxic to all three cell lines despite different extent. Morpfiofogicalfy compared with the control in which cells grow free from drugs, LoVo cells, 72 hours after 5-FU treatment at 8 n M or 64 u M, become karyopyknostic, round, refrangibile, and
decrease in number. In SW480 cells and SW1116 cells.however, they showed less cytotoxicity than LoVo cells. &ff vialnGty assay in exposure to 5-FU also suggested that 5-FU be cytotoxic to all three cell lines. At 12h, 48h and 120h, LoVo was most sensitive to low concentrations of 5-FU (<4 u mol/L), SW480 was least sensitive, and SW1116 was intermediate. At 120h, the ICso of LoVo (0.8 u mol/L) was significantly lower than those for SW480 and SW1116 (2.2 y mol/L and 1.9 u mol/L, respectively, p=0.000). Time-effect curves indicated that survival rates of the three cell lines all decreased with time. But LoVo is most sensitive and SW480 least at lower concentrations of 5-FU(<4 u mol/L, p<0.05). Analyses of the sensitivity and responsiveness of LoVo, SW480 and SW1116 cells to CDDP and
对结直肠癌的治疗方面，目前仍然采用手术、化疗和放疗相结合的综合治疗手段，所用的化疗方案主要是以5-氟尿嘧啶(5-FU)为基础的多个药物组合，临床上实践证明化疗能杀灭术前或术后发生的微转移灶，从而有效地降低进展期病人死亡率，但仍然离我们的期望甚远，目前报道的以5-FU为基础的各种化疗方案的有效率基本上在15～21％之间，可能与低有效率相关的生化机制主要涉及六大类化疗敏感性相关因子，包括药物外排，灭活、损伤DNA的修复，药物作用靶点的调节和其它(如凋亡，端粒长度和端粒酶活性等)。此外，临床回顾性研究还发现大约半数Dukes’C期的CRC病人并不真正需要化疗，而约35％的化疗病人并没有真正得益于系统性化疗。由此表明，对化疗预后或疗效预测分子标记的研究深具意义，它不仅可以提供CRC化疗病人预后信息，还为特定病例化疗与否，化疗方案的选择，即“个体化化疗”提供依据，随着生物芯片技术的发展，这方面的工作愈加显得重要。
顺铂(CDDP)是一种铂类衍生物，它在肺癌、卵巢癌等化疗中被广泛使用。目前在一些医院的肿瘤科与5-FU一起用于CRC病人化疗。由于这两种药物作用机制完全不同，因此被认为能够在联合用药中起到协同作用而提高疗效。但是，
CDDP临床耐药报道较多．临床疗效差异牲极大．因此推测可能存在明确的化疗
敏感相矢机制。
为了寻找与结肠扬细胞系5千U和／或CDDP化疗敏脓牲相矢因子，探讨5－FU
和／或CDDP化疗敏成牲生化机制．我们在此研究了三个人结肠癌细胞系对5－FU
和／或CDDP反应表型、在5－FU和／或CDDP作用下细胞局期分布、胞苦酸台成酶
（TS）mRNA水平、P53、错配修复蛋白hMSHZ、hMLHI表达状况．以及十个与细
胞增殖或死亡相矢的微卫皇位点状况。通过比较药物欧感牲表型和上述遗传背景
之间的夫系，以揭示或部分阐明CRC病人预后或预测分子标记在用药优化和预后
评价中的作用。
第一韶分
三个人结＆＄细胞系对SFU和l或 CDDP处理的敏感性表型及 P53蛋白表达
在这个部分我们首先通过形态学观寨、MTT ＃活牲测定和硫式细胞分析仪检
测了结肠癌细胞系LOVO、SW480和SWill6细胞对5下U和／或CDDP处理的反应．
周期阻滞倩况；并用免疫细胞化学方法检测P53蛋臼在这三个细胞系中的表达倩
况。
结果发现5干U和CDDP M三个细胞系都表现出不同程度的毒牲作用。形态学
观察表明 LOVO细胞在 8 P M或 64 P M的 5－FU作用下出现明显的毒牲反应：核固络、
变圆、折光牲增加．以及细胞数目明显减少；而SW480和SWill6细胞的毒枉反
应则相对较轻些。细胞活力分析也表明．刘低剂云（＜4厂M）处理肘．三个细胞
系中L。Vo细胞刘5·FU最敏感．SW480细胞最不敏感．SWI 116介于两者之间。在
8 P M时．也表现为 LOVo最敏感。在处理后 12小肘，LOVO细胞的 5－FU IC。。值明显
低于其它两个细胞系（分别为：0二2 p M、2．2 u M、1．9 u M，P二0刀加）。肘间－反应曲
线提示．三个细胞系对5－FU的反应有时间依赖性．但LoVO细胞仍然投另两个细
胞系敏感。研究表明SW480和LoVo细胞对CDDP的反应诌具有一定敏感牲．但前
者略比后者欧感．而SWill6则对CDDP耐受。三个细胞系对5－FU＋CDDP的联台应
用都没有表现出协同作用。
细胞周期分析表明．在5－FU作用下．LOVO细胞系出现亚二倍体峰时间比SW480
细胞系提前48小阿；而CDDP作用48小肘后，SW480细胞系比L。V。细胞系出现
更多的亚二倍体降细胞，作用96小肘后，前者亚二倍体蟹细胞数目接近
xi
后者的 2倍（分别为 55．1％和 28．5％）；当两种药物联台使用肘．亚二倍体降细胞
数目明显凉少。此外．两个纫胞系在5－FU作用下诌表现为GIM阻滞，而S M细
胞数目纫下降：但CDDP的作用却刚好相反，它主要使细胞阻滞在S期；两药联
台应用肘，细胞周期分布障药物作用肘间延长出现较大的波动。
P53免疫组织化学染色发现，L。VO细胞系细胞出现散在的巢状分布．核染色
细胞数目少于1％；而SW480和SWill6细胞细胞核出现明显的诌一染色，阳性细
胞数目超过98％。
莆二韶分
SFU和／或CDDP作用下三个结肠癌细胞系胸旮酸合成酶（TS）mRAN表达
在第一部分里，我们发现LoVO细胞系比SW480和SWill6细胞系对5－FU更加
欧感，而CDDP不尽相同。不少研究报道．肿瘤细胞对5－FU的欧感牲和脑吝酸台
成酶（TS）表达水平密切相夫。在此．我们也用 RT·PCR方法在肘间?
The last twenty years have witnessed the revolution in our JL understanding of the processes responsible for the carcinogenesis at molecular level, but the problems still exist, or even are growing as the average lifespan in many countries steadily rises. So far, colorectal cancers are usually classified into three subgroups in terms of molecular biology, genetics as well as clinical features: Familial Adenomatous Polyposis (FAP)-associated CRC, Hereditary Non-Polyposis Colon Cancer (HNPCC) and Sporadic Colorectal Cancer (SCRC), each of them has its predominant molecular portrait.
Besides surgery, radiotherapy and biological therapy, chemotherapy is proven effective in reducing mortality for advanced CRC patients by killing micrometastases disseminated at surgery or occult metastases already present at surgery. Among various regimens, combination therapies of 5-fluorouracil (5-FU) with some second-line agents have been the mainstay of adjuvant chemotherapy for CRC patients to date, but regardless of regimens, the outcome of CRC patients is far from satisfaction owing to various biochemical or non-biochemical mechanisms. In various tumors, at least five groups of sensitivity-associated factors have been identified, including drug efflux, detoxification, DMA repair, drug target regulations and other factors as apoptosis, length of telomere, etc. In addition, clinical data suggests that, in retrospect, nearly half of the patients with Dukes' C colorectal caner might not require adjuvant chemotherapy and about 35%
would die even when treated with systemic chemotherapy. This also highlights the need for informative predictive or prognostic markers that will aid identification of patients best treated with surgery alone, those requiring chemotherapy and those who might benefit from aggressive or experimental therapy.
To elucidate the possible biochemical mechanisms of chemosensitivity, and to identify possible factors involved in response to 5-FU-based regimens in CRC, we studied the chemosensitive phenotype to 5-FU, CDDP or their combination therapy, alterations of cell cycle distribution and transcription level of TS in exposure to drugs, expression of several proteins and the microsatellite status in three human colon cancer cell lines. By comparing the phenotype and their genetic background, we look forward to revealing whether these molecular events can be used as convenient predictors for drug-optimization or prognosis-assessment in CRC patients.
Part I
Phenotype of chemosensitivity to 5-fluorouracil & cisplatin and p53 expression in three human colon cancer cell lines
Firstly, we determined the phenotype of chemosensitivity to 5-fluorouracil and cisplatin in LoVo, SW480 and SW1116 cell lines in vitro, using microscopic morphology, MTT assay and Flow cytometry. Considering that big controversies exist in the biological role of p53 in chemosensitivity, we also analyzed its expression in those three cell lines by immunocytochemistry.
Results showed that 5-FU and CDDP were cytotoxic to all three cell lines despite different extent. Morpfiofogicalfy compared with the control in which cells grow free from drugs, LoVo cells, 72 hours after 5-FU treatment at 8 n M or 64 u M, become karyopyknostic, round, refrangibile, and
decrease in number. In SW480 cells and SW1116 cells.however, they showed less cytotoxicity than LoVo cells. &ff vialnGty assay in exposure to 5-FU also suggested that 5-FU be cytotoxic to all three cell lines. At 12h, 48h and 120h, LoVo was most sensitive to low concentrations of 5-FU (<4 u mol/L), SW480 was least sensitive, and SW1116 was intermediate. At 120h, the ICso of LoVo (0.8 u mol/L) was significantly lower than those for SW480 and SW1116 (2.2 y mol/L and 1.9 u mol/L, respectively, p=0.000). Time-effect curves indicated that survival rates of the three cell lines all decreased with time. But LoVo is most sensitive and SW480 least at lower concentrations of 5-FU(<4 u mol/L, p<0.05). Analyses of the sensitivity and responsiveness of LoVo, SW480 and SW1116 cells to CDDP and
引文
[1]Home page of the National Cancer Institute, U.S.A.
[2]Hardy RG, Meltzer SJ, Jankowski JA. Molecular basis for risk factors. BMJ. 2000;321: 886～9
[3]张建明，柯杨。细胞癌变的遗传模型研究回顾与展望。国外医学遗传学分册。1997；20：39～41
[4]Zingg JM, Jones PA. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. 1997; 18:869～82
[5]Rainier S, F. einberg AP. Genomic imprinting, DNA methyiation, and cancer. 1994; 86:753～9
[6]来茂德．结直肠癌发生的分子机制研究进展。中华病理学杂志。2000；29：450～452
[7]Carethers JM. The cellular and molecular pathogenesis of colorectal cancer.Gastroenterology Clinics of North America. 1996; 25:737～54
[8]Hoops TC, Traber PG.Molecular pathogenesis of colorectal cancer.Hematology/Oncology Clinics of North America. 1997; 11:609～33
[9]Rhyu MS. Molecular mechanisms underlying hereditary nonpolysis colorectal carcinoma. Journal of the National Cancer Institute. 1996; 88:240～51
[10]Service RF. Stalking the start of colon cancer. Science. 1994; 263:1559
[11]Kinzler KVV, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996; 87:159～70
[12] Jen J, Powell SM, Papadopoulos N, et al. Molecular determinants of dysplasia in colorectal lesions. Cancer Research. 1994; 54: 5523-6
[13] Lairmore TC, Norton JA. Advances in molecular genetics. American Journal of Surgery. 1997; 173: 37-41
[14] Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers.Nature. 1998; 396: 643-9
[15] McLeod HL, Murray Gl. Tumor markers of prognosis in colorectal cancer. British Journal of Cancer. 1999; 79: 191-203
[16] Lage H, Dietel M. Involvement of the DNA mismatch repair system in antineoplastic drug resistance. Journal Cancer Research Clinical Oncology. 1999; 125:156-65
[17] Phalman L. Gastroenterology.
[18] Ringborg U, et al. Chemotherapy resistance mechanisms. Acta Oncologica. 1996;35: 76-80
[19] Lehne G, Elonen E, Baekelandt M, et al. Challenging drug resistance in cancer therapy. Acta Oncologica. 1998; 37:431-9
[20] Hansson J. Why some tumors fail to respond to chemotherapy. Acta Oncologica.1998;37:627~8
[21] Pusztai L, Siddik ZH, Mills GB, et al. Physiologic and pathologic drug resistance in ovarian carcinoma. Acta Oncologica. 1998; 37:629-40
[2]Hardy RG, Meltzer SJ, Jankowski JA. Molecular basis for risk factors. BMJ. 2000;321: 886～9
[3]张建明，柯杨。细胞癌变的遗传模型研究回顾与展望。国外医学遗传学分册。1997；20：39～41
[4]Zingg JM, Jones PA. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. 1997; 18:869～82
[5]Rainier S, F. einberg AP. Genomic imprinting, DNA methyiation, and cancer. 1994; 86:753～9
[6]来茂德．结直肠癌发生的分子机制研究进展。中华病理学杂志。2000；29：450～452
[7]Carethers JM. The cellular and molecular pathogenesis of colorectal cancer.Gastroenterology Clinics of North America. 1996; 25:737～54
[8]Hoops TC, Traber PG.Molecular pathogenesis of colorectal cancer.Hematology/Oncology Clinics of North America. 1997; 11:609～33
[9]Rhyu MS. Molecular mechanisms underlying hereditary nonpolysis colorectal carcinoma. Journal of the National Cancer Institute. 1996; 88:240～51
[10]Service RF. Stalking the start of colon cancer. Science. 1994; 263:1559
[11]Kinzler KVV, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996; 87:159～70
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