白血病之分子研究

Molecular Study of Leukemia

劉大智(Ta-Chih Liu)

指導教授 ： 林 勝 豐

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

白血病的癌化分子機轉包括致癌基因與抑癌基因之基因變化逐步累積，另外也與遺傳穩定度及環境因子有關。流行病學研究顯示，環境因子在癌症發生過程佔重要地位，因為每個人對致癌物代謝基因多型性不同，導致個體對白血病的敏感性也不同。另外個體因代謝致癌物並將致癌物清除之去毒能力不同，導致個體罹白血病危險性也不同的。非常多的的特異致癌物代謝基因之基因多型性曾被提出與白血病相關，例如cytochrome 450(CYP)、硫轉移脢(SULT)和麩胺醯基轉硫脢(GST)等。但大部分的研究僅分析單一酵素之基因多型性，並未整合性分析多個相關基因。
最近在腫瘤發生機轉上研究發現，某些正常時不甲基化的抑癌基因發生異常甲基化與癌化有關。而白血病致病機轉和去氧核醣核酸不正常的過度甲基化也被認為相關。在白血病中發現有多個基因不正常過度甲基化。一些基因的過度甲基化在白血病惡化過程中同時被發現，包括p15基因在MDS惡化為急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)的BCR-ABL基因。
在白血病治療上，病人間對化療的反應及毒性出現相當大的差異性，而這些差異則是因個體對化療藥物的代謝基因不同導致。人類藥物代謝基因的基因多型性包括有CYP 3A家族、dihydropyrimidine去氫脢、deoxycytidine激脢、GSTM1、GSTT1、thiopurine甲基轉移脢等。個體中藥物代謝基因之基因多型性將影響個體罹患白血病時的治療效果。
本研究之三個目標為1) 白血病危險因子之探討2) 白血病相關基因甲基化和突變分析 3) 影響治療和預後因子之探討。我們搜集200個急性骨髓性白血病、50個骨髓形成不良症候群 (MDS)、100個急性淋巴性白血病、100個慢性骨髓性白血病包括30個不同期的變化、5株白血病細胞株及300個正常控制組之檢體為材料。首先我們用聚合脢連鎖反應 —限制脢切方法及直接核酸定序來檢測在白血病的致癌物質代謝基因之基因多形性的變化情形。然後利用甲基化特異性聚合脢連鎖反應 (methylation-specific PCR) 來偵測抑癌基因之甲基化及突變。最後我們檢測在白血病的化學治療藥物代謝基因之基因多形性的變化情形。
針對SULT1A1及SULT1A2型多型性基因研究, 我們的結果顯示，病人組中相較於正常控制組其屬SULT1A1*1/*1與SULT1A2*1/*1同型合子基因型佔92.0 %；屬SULT1A1*1/*2與SULT1A2*1/*2異型合子基因型則佔8.0 %。明顯可知在AML檢體中攜帶SULT1A1*1/*2及SULT1A2*1/*2基頻率與正常控制組不同(P=0.04)。白血病與內皮細胞阻斷素(endostatin)之D104N單核苷酸多型性(即第 4349核苷酸由G?A)的關係，結果發現，正常控制組為4349G/G基因型，即形成104D/D同型合子比例為95.5%；為4349G/A基因型，即形成104D/N異型合子佔4.5%；在白血病個案部分，屬4349G /G基因型在126個AML、57個CML、91個ALL個案中所佔比例分別如下：98.4%、94.9%、97.9%；另外，屬4349G /A基因型也分別如下1.6 %、5.1 %、2.1 %；在白血病患者相較於正常人，其內皮細胞阻斷素所攜帶的基因型頻率相當。探討CYP3A5基因多型性跟骨髓性白血病的相關性結果顯示，個案中屬CYP3A5*1/*1基因型在，AML中佔15/188 (8 %)；CML中佔 8/101 (7.9 %)；在MDS中佔3/40 (7.5 %)。而在AML中有88/188 (46.8 %)；CML中有47/101 (46.5 %)；MDS中有20/40 (50 %)是屬於CYP3A5*1/*3基因型。另外，屬CYP3A5*3/*3 基因型，在AML中佔85/188 (45.2 %)；CML中佔46/101 (45.5 %)；MDS中佔17/40 (42.5%)。而攜帶CYP3A5*3對偶基因在白血病個案及正常控制組中頻率相同。從本研究中可知內皮細胞阻斷素基因多型性CYP3A5基因多型性與罹患白血病無關。而台灣人的SULT1A1及SULT1A2基因多型性與罹患急性骨髓性白血病危險性有關。
腫瘤抑制基因PTEN/MMAC1基因, BCL10基因於急性骨髓性白血病之分析, AML檢體出現PTEN/MMAC1基因異常轉錄本(transcript)佔24 %(15/62)；細胞株中佔80 %(4/5)；正常控制組中佔13 %(4/30)。在本研究中全部急性骨髓性白血病個案並沒有LOH或突變情形。出現RNA因不正常裁剪形成異常轉錄本現象，其可以由全部的檢體都同時出現異常轉錄本及正常長度轉錄本結果可證。BCL 10基因於AML個案與正常控制組中其整個轉錄區段中的外顯子(exon)1及3.2有SSCP變異。直接定序結果顯示，在第五個密碼子的第三個鹼基G/T鹼基, 第八個密碼子的第三個鹼基G/A 鹼基, 第213個密碼子的第二個鹼基G/A多型性現象，可見於 AML及正常控制組且其頻率相當。且無論是AML或是細胞株中都沒有發現基因突變現象。我們的結果指出，PTEN/MMAC1 基因可能只在少部分急性骨髓性白血個案佔一重要角色。而BCL 10基因在急性骨髓性白血病中並非主要腫瘤抑制基因。
SOCS1基因突變情形、啟動子CpG島甲基化程度及SOCS1蛋白表現。結果顯示，慢性骨髓性白血病個案檢體其SOCS1基因序列並沒有突變。但在慢性骨髓性白血病芽細胞危象期檢體則有67 %出現高度甲基化情形(p<0.0001)；慢性期檢體則為46 %。然而我們並沒有在正常控制組及慢性骨髓性白血病分子緩解期(remission)檢體中發現SOCS1基因啟動子區段甲基化情形。顯示慢性骨髓性白血病中SOCS1基因因啟動子區段CpG島被甲基化，而造成SOCS1基因不表現(slience)，而於分子緩解期時則出現去甲基化情形而回復SOCS1基因表現。既然SOCS1對多種細胞激素來說是一個廣泛的負向調控蛋白，如果因SOCS1基因不表現之緣故，失去了這種對細胞激素負向調控機制，很有可能在慢性骨髓性白血病病程中扮演重要角色。

關鍵字

白血病 ； 硫酸酯轉移酶1A1 ； 1A2基因 ； CYP3A5基因 ； PTEN/MMAC1基因 ； BCL10基因 ； SOCS1基因 ； endostatin 基因

並列摘要

The molecular pathogenesis of different kinds of cancer including leukemia involves a stepwise accumulation of mutations, affecting both cellular oncogenes and tumor suppressor genes. In our previously studies of FHIT and TSG101tumor suppressor genes in acute myeloid leukemia (AML), we found that the aberrant transcript occurred more frequently in the AML and leukemic cell lines than the normal control. However, no mutations were found in these compared genomic DNA. However, the leukemia formation is a much more complicated process which is associated with genetic instability and environment factors. Epidemiological studies suggest that the environment may play a role in the development of cancers. The genetic differences in the metabolism of carcinogens associated with different susceptibilities to develop leukemia and other cancers have been explored. People are different in their ability to metabolize carcinogens and hence to detoxify chemicals, leading to different risk in getting leukemia. Numerous specific genetic polymorphisms in the cytochrome 450 (CYP), sulphotransferase (SULT) enzyme, and glutathione S-transferase (GST) have been described in leukemia.
Recent studies showed that the abnormal methylation of normally unmethylated CpG island of the promoters of defined tumor suppressor genes is associated with the human cancer development. Abnormal hypermethylation of several genes have been noted in leukemia.
It is also well known that the chemotherapeutic agents for leukemia exhibit wide inter-patient variability in efficacy and toxicity. These inter-patient differences are due to polymorphisms in genes encoding drug metabolizing enzymes and drug targets. Though the inter-individual differences of drug metabolizing enzymes are typically monogenic traits, the overall pharmacological effects of chemotherapy of leukemia are more likely polygenic traits, determined by numerous genes encoding different enzymes involved in multiple pathways. The genetic polymorphisms of human drug metabolizing enzymes include CYP 3A subfamily, dihydropyrimidine dehydrogenase, deoxycytidine kinase, GSTM1, GSTT1, and thiopurine methyltransferase etc. There are several studies demonstrate that the genetic polymorphism of individual drug metabolizing enzyme will influence the treatment result of different kinds of cancers.
We studied CYP3A4, 3A5 gene, SULT1A1 and 1A2, and endostatin polymorphisms in different types of leukemia. The bone marrow and/or peripheral blood from AML, ALL, CML, and MDS were analyzed by a PCR-RFLP and direct sequencing assay. Our data showed that the frequencies of SULT1A1*1/*2 and SULT1A2*1/*2 in AML patients were significantly different to the controls (P=0.04).
The data of CYP3A5 polymorphisms showed that 15/188 (8%), 8/101 (7.9%), and 3/40 (7.5%) of the patients (i.e., 188 AML, 101 CML, 40 MDS) were CYP3A5*1/*1; 46.8%, 46.5%, and 50% were CYP3A5*1/*3; and 45.2%, 45.5%, and 42.5% carried the CYP3A5*3/*3 genotype, respectively. Similar frequencies of the CYP3A5*3 were observed in the leukemic patients and normal controls. Our data of endostatin polymorphisms D104N (nucleotide 4349G?A) showed that 4349G allele in 178 normal Taiwanese was 98% (348/356) and the 4349A allele frequency was 2% (8/356). The frequencies of homozygous of 4349G (104D/D) and heterozygous of 4349G/A (104D/N) were 95.5% and 4.5% (8/178), respectively. Among those patients of 126 AML, 57 CML, and 91 ALL, 124/126 (98.4%), 55/57 (94.9%), and 89/91 (97.9%), respectively, were 4349G /G. In addition, 2/126 (1.6 %), 2/57 (5.1 %), and 2/91 (2.1 %), respectively were 4349G /A. Similar frequencies of endostatin polymorphisms were observed in leukemic patients and normal controls. Consequently, the findings of SULT1A1 and SULT1A2 polymorphisms in Taiwanese were associated with the higher risk of AML. However, the CYP3A5 and the endostatin p polymorphism were not associated with the risk of myeloid leukemia.
The analysis of PTEN/MMAC1gene and BCL 10 gene in 62 AML patients, 5 hemopoietic cell lines (HL60, U937, Raji, KG-1, K562), and 30 normal controls showed aberrant PTEN/MMAC1 24% AML patients, 80% cell lines, and 13% normal controls. Loss of heterozygosity (LOH) analysis and PCR-SSCP of the entire coding region showed that none of the AML cases had LOH or mutation. BCL 10 gene analysis showed that AML cases and normal controls had SSCP variants in exon 1 and exon 3.2. Direct sequencing polymorphism of G/T at the third base of codon 5, G/C at the third base of codon 8, and G/A at the second base of codon 213 showed that found both in AML and normal controls with similar frequencies. No pathogenic mutations were found in any of the AML cases or cell lines. Our data showed that transcripts containing deletions or insertions could be frequently detected, but also noted in RNA from normal tissues. As we could not detect the deletions in genomic DNA, it seems that aberrant splicing may be the underlined mechanism for the truncated transcripts detected. The possibility of the aberrant transcripts caused by pre-mRNA splicing that was less stringent in cancer cells than in normal tissues should be considered. Our results indicate that the PTEN/ MMAC1 gene may play a role in a small percentage of AML, and the BCL 10 gene is not the target tumour suppressor gene in AML.
The mutations analyses, CpG island methylation status, and the expression of the SOCS1 gene in 112 CML samples, 5 leukemia cell lines, and 30 normal controls showed no genetic mutations of SOCS1 gene. We found that SOCS1 gene in 67% and 46% of the blastic and chronic phase of CML, respectively, was hypermethylated (p<0.0001). However, there were no methylation of the SOCS1 gene in normal controls or CML in molecular remission. The methylation status of the SOCS1 gene is consistent with the results of the real-time quantitative RT-PCR and immunocytochemistry staining. Our results demonstrate that the SOCS1 gene silencing is caused by the methylation of CpG islands in CML and reversed to unmethylation status in molecular remission. Since the SOCS1 has a universe activity to negatively regulate several cytokine signaling pathways, the loss of the negative regulation of cytokine signaling by the SOCS1 may play a role in the pathogenesis of CML progression.

並列關鍵字

leukemia ； SULT1A1 ； 1A2 gene ； CYP3A5 gene ； PTEN/MMAC1gene ； BCL10 gene ； SOCS1gene ； endostatin gene

參考文獻

1.Lin PM, Liu TC, Chang JG, et al. Aberrant FHIT transcripts in acute myeloid leukaemia. Br J Haematol. 1997; 99, 612-617.

2.Lin PM, Liu TC, Chang JG, et al. Aberrant TSG 101 transcripts in acute myeloid leukaemia. Br J Haematol. 1998; 102, 753-758.

3.Lin SF, Pai-Mei Lin, Liu TC, et al. Clinical implications of aberrant TSG101 transcripts in acute myeloid leukemia. Leukemia and Lymphoma 2000; 36: 463-466.

4.Wang NM, Chang JG, Liu TC, et al. Aberrant transcripts of FHIT, TSG 101, and PTEN/MMAC1 genes in normal peripheral mononuclear cells. Int J Oncol 2000; 16: 75-80.

5.Wang NM, Chang JG. Are aberrant transcripts of FHIT, TSG 101, and PTEN/MMAC1 oncogenesis related? Int J Mol Med 1999; 3: 491-495.