| Abstract: | FHIT抑癌基因包含一個染色體易斷裂的位置：FRA3B。過去的研究指出，在子宮頸癌細胞株(SiHa)中，人類乳突瘤病毒(HPV) DNA會嵌入FRA3B，而造成其附近的FHIT基因缺失部份的DNA。而本研究室過去在肺腫瘤組織中偵測到HPV 16/18 DNA 的存在，尤其是不抽菸女性有高達七成的感染率。因此本研究欲藉由分析HPV感染，FHIT抑癌基因的基因座缺失(Loss of heterozygosity)和其蛋白表現之相關性，以釐清HPV參與肺腫瘤形成之可能性。本研究共選取174位非小細胞肺癌(non-small cell lung cancer, NSCLC)患者，包括78位抽菸者、34位不抽菸男性患者與62位不抽菸女性患者。首先利用螢光標記的微衛星序列引子：D3S1300、D3S1234和D3S1313來觀察FHIT異質性基因座缺失，結果發現31%的不抽菸女性，39%的不抽菸男性和55%抽菸男性肺癌組織在FHIT抑癌基因發生異質性基因座缺失(LOH)的現象，在不抽菸個案之肺腫瘤組織也有高比例的異質性基因座缺失(LOH)，與過去認為抽菸會有較高比例基因座異質性缺失的研究結果不相同。過去的研究指出HPV感染可能會嵌入宿主染色體造成基因發生LOH，而本實驗室先前的研究也發現不抽菸肺癌患者之HPV感染率高於抽菸之肺癌患者，因此推測不抽菸肺癌患者之腫瘤組織有較高比例之FHIT LOH可能與HPV的感染有關。經以巢疊式聚合鏈鎖反應(nested-PCR)分析HPV感染情形並利用統計分析後發現，在不抽菸肺癌患者中，HPV感染者其FHIT發生LOH的比率高於沒有HPV感染者(HPV infection 45％ vs. non-HPV infection 23％ , P=0.030)。更進一步在不抽菸女性肺癌患者中，發現HPV感染者其FHIT發生LOH的比率高於沒有HPV感染者(HPV infection 44％ vs. non-HPV infection 15％ , P=0.025)。由以上結果推測，在不抽菸患者甚至不抽菸女性患者中，其FHIT基因發生異質性基因座缺失可能是由於HPV感染所致。
此外，為進一步了解HPV感染以及FHIT發生LOH是否會導致蛋白的不表現，本研究利用免疫組織化學染色(immunohistochemistry;IHC)分析FHIT蛋白表現的情形，結果發現在不抽菸女性肺癌患者，HPV感染對於FHIT蛋白表現，並無直接的影響。而利用在HPV感染的群體中，發生LOH的肺腫瘤組織相較於不發生LOH者，其有較高的比率FHIT蛋白較不表現，但不達到統計上的差異。經由以上的結果，推測HPV感染的肺腫瘤組織可能會經由FHIT基因發生LOH，促使其蛋白較不表現的情形。
The fragile histidine triad (FHIT) gene encompasses the common chromosome fragile site FRA3B. Human papilloma virus (HPV) has been found to be able to integrate its genes into the chromosome 3 fragile site of cultured cells, deleting a piece of DNA which includes the FHIT gene. Previous report indicated that human papillomavirus 16/18 (HPV) was associated with lung cancer incidence in Taiwanese nonsmoking women. In this study, we examined FHIT gene LOH and its protein expression may be helpful to verify whether HPV infection was involved in lung tumorigenesis.
174 lung tumors were enrolled in this study including 78 smoking male, 33 nonsmoking male and 63 nonsmoking female. FHIT gene LOH was detected by polymerase chain reaction (PCR) using fluorescently labelled intragenic microsatellite markers D3S1300, D3S1234, and D3S1313 and then PCR products were loaded on a DNA sequencer(ABI PRISM 310) for allele loss analysis. The presence of HPV 16/18 DNA was detected by PCR using type-specific primers. FHIT protein expression in lung tumors was detected by immunohistochemistry using by polyclonal antibody anti-FHIT (Zymed,laboratories Inc.). Data were statistically analyzed by SPSS-PC software.
In this study, 32% of nonsmoking female, 39% of nonsmoking males and 55% smoking male lung cancer patients had FHIT LOH positive. This result was consistent with previous reports showing that FHIT LOH was correlated with cigarette smoking. To understand why more FHIT LOH was observed in nonsmoking cases, we suspect HPV infection may be involved in the increase of FHIT LOH in nonsmoking cases because HPV infection was frequently found in nonsmoking lung cancer patients. Our data showed that FHIT LOH frequency in nonsmoking tumors with HPV16/18 was peaked to 45% (21 of 47) compared to those without HPV16/18 infection (23%, 11 of 47, P = 0.030). And then in nonsmoking female tumors with HPV16/18 was peaked to 44% (15 of 34) compared to those without HPV16/18 infection (15%, 4 of 26, P = 0.025). In addition, immunohistochemistry data showed that more frequent FHIT protein negative immunostainings (32%) was observed in nonsmoking female tumors with FHIT LOH compared to those without FHIT LOH (12%,5 of 41,P=0.086).And then my study find that HPV infection was not assosciated with FHIT protein expression.My study find that more frequent FHIT protein negative immunostainings (27%) was observed in FHIT LOH and HPV both positive lung tumors compared with no-FHIT LOH but HPV infection lung tumors (11%)(P = 0.370).
These results strongly suggest that HPV16/18 infection involved in lung tumorgenesis, may be at least in part mediated through increased FHIT LOH frequencies to cause FHIT gene inactivation. |
