扁平細胞癌抗原在子宮頸癌上功能及基因治療的研究

行政院國家科學委員會專題研究計畫 成果報告

扁平細胞癌抗原在子宮頸癌上功能及基因治療的研究

中 華 民 國 92 年 10 月 29 日

Introduction: 子宮頸癌在台灣為婦女主要的癌症死亡原因之一。臨床上我們用血清中扁平 上皮細胞癌抗原的濃度高低來預測子宮頸癌病人的預後以及作為腫瘤標誌 (tumor marker) 來追蹤病人的病情。我們先前的臨床研究發現，子宮頸癌手術 前的扁平上皮細胞癌抗原的高低可以用來預測術後病人是否要接受輔助性化學 治療或是放射治療。即使如此，扁平上皮細胞癌抗原在子宮上皮細胞所扮演的角 色仍然不詳。扁平上皮細胞癌抗原分為 SCCA1及 SCCA2，在我們初步用 RT-PCR 的 方法分析正常子宮頸上皮，HPV-immotalized cells 如 Z172，183 等及癌細胞株 以及人類子宮頸癌組織發現，在 HPV-immotalized cells，Z172，183 均沒有扁 平上皮細胞癌抗原的表現。此意味著在子宮上皮細胞癌化初始階段，扁平上皮細 胞癌抗原的表現是被抑制的。在癌化成癌細胞後，扁平上皮細胞癌抗原（主要是 SCCA2）才又表現出來。 Results

we have checked SCC antigens expressions in normal uterine cervical cells and carcinoma tissue by one-step RT-PCR. Total RNA were isolated by RNeasy Mini Kit. After annealing 5 mg of total RNA with oligo(dT), cDNA was prepared with Superscript II containing Moloney Murine leukemia Virus Reverse Transcriptase (Life technologies, Inc., Gainthersbrg, MD).. The sequences of SCCA1 sense primer and antisense primer were 5’-AGGTAATATTGCAGCAAT-3’, and 5’-GTAGGTGATGATCCGAATCC-3’. The sequences of SCC A2 sense primer and antisense primer were 5’-TGGGACTATTGGCAATGAT-3’ and 5’-GGAGATGATATAATTCGACTAG-3’ for targeting 576 base paire product of both SCC A1 and SCCA2. The RT-PCT results were shown below. The sequences of GAPDH sense and antisense primer were 5’-TGGCGTCTTCACCACCAT-3’ and 5’-CACCACCCTGTTGCTGTA-3’ as control.

( I ) SCC antigen expression in different human tissues.

Figure 1:

SCC A1 and A2 expression by RT-PCR from three normal patient’s cervix epitheliums. The SKG-IIIb cells as SCC A1 control.

N1 N2 N3 SKG-IIIb 576 bp

576 bp

500bp

200bp

576bp

SCCA1 cut by EcoRv

SCCA2 was not cut by EcoRv

In normal uterine cervical epithelium cell, SCC A1 expression is predominantly, compared with SCC A2 expression. However, in cervical cancer tissue SCC A2 expression is higher than SCC A1 (Fig. 2) We have checked 4 cases with cervical carcinomas as following:

C: control, in SCC A1, we use SKG-IIIb cells. In SCCA2, we use cancer cell line HT-3 as control.

Figure 2:

Case 1 and 3 were adenocarcinoma cases, so no expression of SCC antigen was noted. In case 2, 4, there were a elevated SCC A2 expression level than SCC A1.

Further, we tested other organs in female pelvis. Meanwhile , we can not find expression of SCC antigen in uterus as well as ovary. C: control, 1. myometrium, 2.endometrium, 3. fallopian tube, 4. Ovary

C 1 2 3 4

SCC A1

SCC A2

GAPDH

C 1 2 3 4

Case 1 Case 2 Case3

SCC A1 SCC A2 GAPDH

We also clone SCC A2 promter for part of gene therapy in our lab. To test the expression, we constructed the promter to a promoterless plasmid pEGFP and transfect to HT-3 cell. The result was as following picture (Fig. 3). That showed the gene we cloned was functional.

Figure 3. pEGFP-SCCA2 promoter in HT-3 cells (x 200).

For further defined the SCC A2 promoter is specific in SCC A2 expression cancer cell we transfected th pEGFP-SCC A2 promter into different cells, including HT-3, SKG-IIIb, and normal cervical cells. The results were as following. The plasmid only expressed in SCC A2 expressed HT-3 cervical cancer cell which is expressed SCC A2.

Figure 4.

All adenoviruses have linear double-stranded DNA genomes of approximately 38 HT-3 SKG-IIIb Normal cervical cell

kb. They are common causes of upper respiratory diseases in human. The viral capsid is non-envoloped and is composed of the structural proteins, hexon, penton which bind the aV b3 and aVb5 intergrins to allow virus internalization, and fiber which binds the coxsacki and adenovirus receptor, CAR . To test the infection susceptibility of cervical cancer cell line as well as normal cervical epithelial cells, we infectwed the cells with adenovirus with Lac. To our surprise, the results showed cancer cervical caner cells were much easier to be infected by adenovirus then normal ones. That let us with confidence to use this vector to treat cervical cancer in gene therapy. The results were showed in figure 5.

J82 and T24 are bladder cancer cells used as positive and negative control respectively. Normal, normal cervical epithelium cells; Cx, SiHa, HeLa, HT-3, Caski all are cervical cancer cells.

Figure 5.

Conclusion:

Using SCC antigen promoter gene as gene therapy seems to be a good strategy in treating uterine cervical cancer patients.

Ad/Lac Z in cell lines

J82 T24 SiHa HeLa HT3 Normal Cx Caski 0 10 20 30 40 50 60 70 MOI 100 MOI 10 Percentage of Infection (%)