Cell Biochem Biophys DOI 10.1007/s12013-014-9989-1
ORIGINAL PAPER
Expression of the HPV18E2 Gene in Cervical Cancer and Premalignant Lesions and its Clinical Significance Ling Mou • Bin Wu • Ya Hu • Ying Lan Feng-Lin Lv
•
Ó Springer Science+Business Media New York 2014
Abstract This study aims to observe the expression of the HPV18E2 gene in cervical cancer and premalignant lesions and to investigate its clinical significance. The expression of the HPV18E2 gene in the cervical tissues obtained from 38 women with cervical lesions was detected using the RT-PCR method. The pathological changes were graded based on cervical intraepithelial neoplasia (CIN) criteria. The HPV18E2 gene was expressed mainly in cervical premalignant lesions, 60 % in Grade I CIN, 33.3 % in Grade II CIN, and 28.6 % in Grade III CIN. No expression was detected in cervical cancer and chronic cervical inflammation. This study suggests that peptides vaccine targeting the HPV18E2 protein may disrupt and prohibit the progress of diseases induced by HPV 18 infection (i.e., CIN and cervical cancer). Keywords Cervical Cancer Gene HPV18E2 Premalignant Lesions
Currently, the target proteins of the anti-HPV 18 vaccine are mainly the early proteins E6 and E7. The vaccine has also been proven to be effective to some extent. However, recent research has found that the immune response of the human body to the E6 and E7 proteins of high-risk HPV 16 and 18 is not related to the spontaneous regression of lesion, which might be explained by the high expression of E6 and E7 in late-stage lesion tissue and the inhibition of their expression by the E2 protein at the early stage of infection and cervical intraepithelial neoplasia (CIN) [4]. On the contrary, the E2 protein is stably expressed at the early stage of infection and CIN; the high expression rate of this major regulating protein is essential for viral DNA replication and gene expression [5]. Therefore, we hypothesize that the conditions caused by HPV 18 can be halted at the early stage using HPV18E2 as the target protein. Our department used specific CTL (Cytotoxic T lymphocyte, CTL) epitopes of E2 as candidate vaccine to investigate the expression of the HPV18E2 in cervical cancer tissues and precancerous lesions, which may be used as base for a novel vaccine for cervical cancer.
Introduction Cervical cancer is one of the most common malignant tumors that affect women, the incidence of which is closely related to human papilloma virus (HPV) infection, especially HPV 16 and 18 [1–3]. L. Mou Y. Hu Y. Lan Department of Gynecology and Obstetrics, 324th Hospital of PLA, Chongqing 400020, People’s Republic of China L. Mou B. Wu F.-L. Lv (&) College of Bioengineering and Key Laboratory of Biorheological Science and Technology, Chongqing University, Chongqing 400044, People’s Republic of China e-mail: [email protected]
Materials and Methods Patients Thirty-nine cervical tissue samples were collected from outand inpatients of our hospital between January and December 2005. The median age of the patients was 36.0 ± 0.9 years, and the age range was 21–67 years. After cytological screening, all the patients underwent colposcopy, loop electrosurgical excision procedure, and uterectomy, and the biopsy samples were diagnosed with pathological examination. Based on cervical pathological diagnostic criteria [6], CIN was classified as either Grade I,
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Cell Biochem Biophys
II, or III. Grade I is characterized as having slight atypical hyperplasia; Grade II is medium atypical hyperplasia; and Grade III is severe atypical hyperplasia. Eight patients had chronic inflammation, ten had Grade I CIN, nine had Grade II CIN, seven had Grade III CIN, and five had cervical cancer. This investigation was approved by medical ethics committee of Da Ping Hospital of the Third Military Medical University, and consent forms were signed by the patients. Major Agents and Instruments Micro-high-speed refrigerated centrifuge (Beckman Ltd, USA); MJ. Research PCR Machine (PE Ltd, USA); Gel image analysis system (Bio-Rad Ltd, USA); FX2302 Camera Obscura–UV detector (Shanghai Fuxing High Technology Ltd). RT-PCR Kit (Omega Ltd, USA); 2000 Marker: molecular weight sequentially is 2000, 1000, 750, 500, 250, 100 bp (Dalian TaKaRa Co.). Primer Design HP18E2 Primer: Forward Primer: 50 2 CGG AAT TCA TGG A GA CTC TTT GCC230 ; backward primer: 50 2 CGG AAT TCA. TGC AGA CAC CGA A GC230 ; b2actin primer: Forward primer: 50 2. TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA230 ; backward primer: 50 2 CTA GAA GCA TTT GCG GTG GAC GAT GGA. GGG230 . The above primers were synthesized by Shanghai Bioengineering Ltd.
The RNA was dissolved in 10–35 lL DEPC(Diethyl pyrocarbonate) water (stored in liquid nitrogen or lowtemperature refrigerator). A denaturing RNA electrophoresis was run to observe the 18 and 28 s bands. A 260/280 absorbance was tested, and the concentration of RNA was calculated. For the RT reverse transcription reaction, 1 lL dNTPs, 1 lL MmLV, 4 lL 5 9 MmLV reverse transcription reaction buffer, 1 lL Olig (dT) 16, and 0.4 lL Rnasin were mixed well. An appropriate amount of RNA sample (about 0.5–2 lg) was added for a final volume of 20 lL with ddH2O. The solution was mixed well and incubated in water bath for 2 h (37 or 42 C), then thermally denatured for 5 min at 95 C and used immediately (or stored at -20 C for later use). For PCR, we follow the instructions in the RT-PCR kit. The details were as follows: 30 cycles at 94 C for 2 min, then 94 C for 50 s, 54 C for 30 s, 72 C for 2 min, and an extension at 72 C for 10 min. The reaction product was stored at 4 C. Four microliters of the reaction product were analyzed using 1.5 % agarose gel electrophoresis.
Results As shown in Fig. 1, the HPV18E2 band showed at 1000 BP in the electrophoresis analysis. The results were statistically analyzed and showed the following: E2 had the highest expression rate in Grade I CIN (60 %), while the expression rates in Grades II and III CIN were 33.3 % and 28.6 %, respectively. However, no
RT-PCR Test To extract RNA, 50–100 mg of the tissue samples were frozen with liquid nitrogen, ground to powder form, and mixed with 1 mL Trizol in a glass homogenate. The mixture was transferred into a new centrifuge tube and mixed with 5 mL syringe. Then the mixture was incubated on ice for 5 min. After centrifugation for 5 min (12,000 g, 4 C), the supernatant was transferred into a new 1.5-mL tube. Chloroform (0.2 mL) was added, and the mixture was incubated on ice for 5 min after shaking. The mixture was again centrifuged for 10 min (\12,000 g, 4 C), and the upper liquid layer was transferred into a new 1.5-mL tube. The mixture was shaken after 0.5 mL isopropyl acetone was added and incubated on ice for 5 min. The mixture was again centrifuged for 5 min (\12,000 g, 4 C), and the upper supernatant layer was discarded. Ethanol (1 mL) was added, and the mixture was shaken well. Afterwards, the mixture was again centrifuged for 5 min (\ 7500 g, 4 C), and the upper supernatant layer was discarded. The mixture was allowed to become transparent at room temperature.
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Fig. 1 Expression of HPV18E2
Cell Biochem Biophys Table 1 Statistics of HPV18E2 expression Group
n
Positive (n)
Expression of E2 (%)
Chronic inflammation
8
0
0
CIN I
10
6
600
CIN II
9
3
30
CIN III
7
2
28.6
2
0
0
Cervical cancer stage Ia
1
0
0
Cervical cancer stage IIb
2
0
0
Carcinoma in situ of the cervix
expression of E2 was found in the cancer tissue or chronic inflammation tissue (Table 1).
Discussion The coding gene sequence of the HPV18E2 protein was located in the 2817–3914 position of the HPV genome. This protein consists of 365 amino acids [7]. E2 is a transactivator involved in the transcription of viral DNA and can delay the mitosis of chromosome. In malignant tumor relevant to HPV, viral DNA often integrates in host chromosome. However, this kind of integration is rarely found in precancerous lesion or CIN. Only integrated HPV has malignant transformation capacity, because HPV-DNA controls the genetic product of the host. Therefore, integration is generally thought to be an activation mechanism of cancer from precancerous lesion. HPV 16 and 18 are two major types of cervical cancer–causing virus. A previous study has found that HPV 18 infection was more common in adenocarcinoma and adenosquamous carcinoma [8], with higher chance of pelvic lymph node metastasis and interstitial infiltration and higher recurrence rate. The integration of the HPV18 gene into the host cell is critical for cancer development. This process involves the encoding of the two proteins E1 and E2. Im et al. [9] found that E2 can bind to early promoter of HPV 18 to inhibit the transcription of E6 and E7. After a virulent form of HPV infects the cervical epithelium, the viral DNA integrates into the host chromosome and causes the continuous expression of the E6 and E7, which is the main mode by which cervical cancer develops [10]. Therefore, loss of E2 expression causes the activation of the cancer gene. In cell strains infected with HPV 18, E2 is inactivated, which indicates that the E2 inhibits the progress of cancer. Ectopic expression of the E2 in cervical cancer strains strongly inhibits the proliferation of cells. Introducing E2 into cancer cells infected with HPV can inhibit cell growth, hold the cell in the G1 phase, and promote cell aging and apoptosis. This is an intrinsic function of the E2, which is independent of the cancer-inhibiting proteins E6 and E7 [11].
In addition, the E2 induces apoptosis in HPV-negative cells. In HeLa cells, after E6 is inhibited by E2, the P53 gene is stabilized, and the P21 which is a negative regulating gene of cell cycle is activated, which holds the cell in the G1 phase. Inhibition of the E7 causes cell aging via the pRb pathway. Research has shown that E2 had highest expression at CIN stage while the E2 expression was lost in invasive cervical cancer tissue [12]. In the 30 cases included in this study, E2 expression is detected via RTPCR, and the results showed that E2 had a highest expression rate in Grade I CIN (60 %); expression rates in Grades II and III CIN, meanwhile, were 33.3 and 28.6 %, respectively. However, no expression of E2 was found in cancer tissue or chronic inflammation tissue. This is because during late-stage CIN or in highly malignant cervical cancer, E2 gene is broken during linear integration of the viral DNA into the genome. Therefore, we propose that the lesion tends to transform into malignant cancer when no E2 expression is detected during the early stage of cervical lesion. We believe that the E2 protein can be used as the target protein for novel therapeutic options. From the perspective of immune intervention, the therapeutic target proteins for late-stage CIN and highly malignant cervical cancer should be the E6 and E7, because both are highly expressed in the lesion tissue and necessary for cells to stay in conversion status. However, during early-stage infection or low malignant CIN, the E2 protein is the main component of the immune system because it is highly expressed at this stage. It can also supplement the expressions of the E6 and E7 proteins. Research has found that in cottontail rabbit papillomavirus (CRPV) model, cellular response to E2 is relevant to the regression of HPV lesion [13]. Clinical evidence has also proved that elevated level of Th cell (specific to the COOH end of the E2 protein) can be detected in women diagnosed with CIN with HPV 18 infection during the regression of lesion. This indicates that the cellular immunological responses to the E2 protein can help remove the virus. If a vaccine specific to HPV18E2 can be developed, then the lesion caused by HPV 18 will be halted at the stage of CIN and highly malignant lesion will be removed before its transformation into cancer, which will effectively lower the incidence of cervical cancer.
References 1. Bray, F., Sankila, R., Ferlay, J., et al. (2002). Estimates of cancer incidence and mortality in Europe in 1995. European Journal of Cancer, 38, 99–166. 2. Vizcaino, A. P., Moreno, V., Bosch, F. X., et al. (1998). International trends in the incidence of cervical cancer: i. Adenocarcinoma and adenosquamous cell carcinomas. International Journal of Cancer, 75, 536–545.
123
Cell Biochem Biophys 3. Ma, Y., Yu, C., Leng, Z., et al. (2004). Investigation for the relation between HPV infection and cervical squamous intraepithelial lesion and cervical carcinoma. Chongqing Medical Journal, 33, 1011–1013. 4. Resnick, R. M., Cornelissen, M. T. E., Wright, D. K., et al. (1990). Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. Journal of the National Cancer Institute, 82, 1477–1484. 5. Wang, Y., Li, L., Li, Y., et al. (2006). Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells. Acta Academiae Med Mil Tertiae, 28, 1280–1282. 6. Clifford, G. M., Smith, J. S., Aguado, T., et al. (2003). Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. British Journal of Cancer, 89, 101–105. 7. Albers, A., Abe, K., & Hunt, J. (2005). Antitumor activity of human papillomavirus type 16 E7-specific T cells against virally infected squamous cell carcinoma of the head and neck. Cancer Research, 65, 11146–11155. 8. Thierry, F., Benotmane, M. A., Demeret, C., et al. (2004). A genomic approach reveals a novel mitotic pathway in papillomavirus carcinogenesis. Cancer Research, 64, 895–903.
123
9. Im, S. S., Wilczynski, S. P., Burger, R. A., et al. (2003). Early stage cervical cancers containing human papillomavirus type 18 DNA have more nodal metastasis and deeper stromal invasion. Clinical Cancer Research, 9, 4145–4150. 10. Francis, D. A., Schmid, S. I., & Howley, P. M. (2000). Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells. Journal of Virology, 74, 2679–2686. 11. DeFilippis, R. A., Goodwin, E. C., Wu, L., et al. (2003). Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. Journal of Virology, 77, 1551–1563. 12. Demeret, C., Garcia-Carranca, A., & Thierry, F. (2003). Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein. Oncogene, 22, 168–175. 13. Blachon, S., Bellanger, S., & Demeret, C. (2005). Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis. Journal of Biological Chemistry, 280, 36088–36098.
ORIGINAL PAPER
Expression of the HPV18E2 Gene in Cervical Cancer and Premalignant Lesions and its Clinical Significance Ling Mou • Bin Wu • Ya Hu • Ying Lan Feng-Lin Lv
•
Ó Springer Science+Business Media New York 2014
Abstract This study aims to observe the expression of the HPV18E2 gene in cervical cancer and premalignant lesions and to investigate its clinical significance. The expression of the HPV18E2 gene in the cervical tissues obtained from 38 women with cervical lesions was detected using the RT-PCR method. The pathological changes were graded based on cervical intraepithelial neoplasia (CIN) criteria. The HPV18E2 gene was expressed mainly in cervical premalignant lesions, 60 % in Grade I CIN, 33.3 % in Grade II CIN, and 28.6 % in Grade III CIN. No expression was detected in cervical cancer and chronic cervical inflammation. This study suggests that peptides vaccine targeting the HPV18E2 protein may disrupt and prohibit the progress of diseases induced by HPV 18 infection (i.e., CIN and cervical cancer). Keywords Cervical Cancer Gene HPV18E2 Premalignant Lesions
Currently, the target proteins of the anti-HPV 18 vaccine are mainly the early proteins E6 and E7. The vaccine has also been proven to be effective to some extent. However, recent research has found that the immune response of the human body to the E6 and E7 proteins of high-risk HPV 16 and 18 is not related to the spontaneous regression of lesion, which might be explained by the high expression of E6 and E7 in late-stage lesion tissue and the inhibition of their expression by the E2 protein at the early stage of infection and cervical intraepithelial neoplasia (CIN) [4]. On the contrary, the E2 protein is stably expressed at the early stage of infection and CIN; the high expression rate of this major regulating protein is essential for viral DNA replication and gene expression [5]. Therefore, we hypothesize that the conditions caused by HPV 18 can be halted at the early stage using HPV18E2 as the target protein. Our department used specific CTL (Cytotoxic T lymphocyte, CTL) epitopes of E2 as candidate vaccine to investigate the expression of the HPV18E2 in cervical cancer tissues and precancerous lesions, which may be used as base for a novel vaccine for cervical cancer.
Introduction Cervical cancer is one of the most common malignant tumors that affect women, the incidence of which is closely related to human papilloma virus (HPV) infection, especially HPV 16 and 18 [1–3]. L. Mou Y. Hu Y. Lan Department of Gynecology and Obstetrics, 324th Hospital of PLA, Chongqing 400020, People’s Republic of China L. Mou B. Wu F.-L. Lv (&) College of Bioengineering and Key Laboratory of Biorheological Science and Technology, Chongqing University, Chongqing 400044, People’s Republic of China e-mail: [email protected]
Materials and Methods Patients Thirty-nine cervical tissue samples were collected from outand inpatients of our hospital between January and December 2005. The median age of the patients was 36.0 ± 0.9 years, and the age range was 21–67 years. After cytological screening, all the patients underwent colposcopy, loop electrosurgical excision procedure, and uterectomy, and the biopsy samples were diagnosed with pathological examination. Based on cervical pathological diagnostic criteria [6], CIN was classified as either Grade I,
123
Cell Biochem Biophys
II, or III. Grade I is characterized as having slight atypical hyperplasia; Grade II is medium atypical hyperplasia; and Grade III is severe atypical hyperplasia. Eight patients had chronic inflammation, ten had Grade I CIN, nine had Grade II CIN, seven had Grade III CIN, and five had cervical cancer. This investigation was approved by medical ethics committee of Da Ping Hospital of the Third Military Medical University, and consent forms were signed by the patients. Major Agents and Instruments Micro-high-speed refrigerated centrifuge (Beckman Ltd, USA); MJ. Research PCR Machine (PE Ltd, USA); Gel image analysis system (Bio-Rad Ltd, USA); FX2302 Camera Obscura–UV detector (Shanghai Fuxing High Technology Ltd). RT-PCR Kit (Omega Ltd, USA); 2000 Marker: molecular weight sequentially is 2000, 1000, 750, 500, 250, 100 bp (Dalian TaKaRa Co.). Primer Design HP18E2 Primer: Forward Primer: 50 2 CGG AAT TCA TGG A GA CTC TTT GCC230 ; backward primer: 50 2 CGG AAT TCA. TGC AGA CAC CGA A GC230 ; b2actin primer: Forward primer: 50 2. TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA230 ; backward primer: 50 2 CTA GAA GCA TTT GCG GTG GAC GAT GGA. GGG230 . The above primers were synthesized by Shanghai Bioengineering Ltd.
The RNA was dissolved in 10–35 lL DEPC(Diethyl pyrocarbonate) water (stored in liquid nitrogen or lowtemperature refrigerator). A denaturing RNA electrophoresis was run to observe the 18 and 28 s bands. A 260/280 absorbance was tested, and the concentration of RNA was calculated. For the RT reverse transcription reaction, 1 lL dNTPs, 1 lL MmLV, 4 lL 5 9 MmLV reverse transcription reaction buffer, 1 lL Olig (dT) 16, and 0.4 lL Rnasin were mixed well. An appropriate amount of RNA sample (about 0.5–2 lg) was added for a final volume of 20 lL with ddH2O. The solution was mixed well and incubated in water bath for 2 h (37 or 42 C), then thermally denatured for 5 min at 95 C and used immediately (or stored at -20 C for later use). For PCR, we follow the instructions in the RT-PCR kit. The details were as follows: 30 cycles at 94 C for 2 min, then 94 C for 50 s, 54 C for 30 s, 72 C for 2 min, and an extension at 72 C for 10 min. The reaction product was stored at 4 C. Four microliters of the reaction product were analyzed using 1.5 % agarose gel electrophoresis.
Results As shown in Fig. 1, the HPV18E2 band showed at 1000 BP in the electrophoresis analysis. The results were statistically analyzed and showed the following: E2 had the highest expression rate in Grade I CIN (60 %), while the expression rates in Grades II and III CIN were 33.3 % and 28.6 %, respectively. However, no
RT-PCR Test To extract RNA, 50–100 mg of the tissue samples were frozen with liquid nitrogen, ground to powder form, and mixed with 1 mL Trizol in a glass homogenate. The mixture was transferred into a new centrifuge tube and mixed with 5 mL syringe. Then the mixture was incubated on ice for 5 min. After centrifugation for 5 min (12,000 g, 4 C), the supernatant was transferred into a new 1.5-mL tube. Chloroform (0.2 mL) was added, and the mixture was incubated on ice for 5 min after shaking. The mixture was again centrifuged for 10 min (\12,000 g, 4 C), and the upper liquid layer was transferred into a new 1.5-mL tube. The mixture was shaken after 0.5 mL isopropyl acetone was added and incubated on ice for 5 min. The mixture was again centrifuged for 5 min (\12,000 g, 4 C), and the upper supernatant layer was discarded. Ethanol (1 mL) was added, and the mixture was shaken well. Afterwards, the mixture was again centrifuged for 5 min (\ 7500 g, 4 C), and the upper supernatant layer was discarded. The mixture was allowed to become transparent at room temperature.
123
Fig. 1 Expression of HPV18E2
Cell Biochem Biophys Table 1 Statistics of HPV18E2 expression Group
n
Positive (n)
Expression of E2 (%)
Chronic inflammation
8
0
0
CIN I
10
6
600
CIN II
9
3
30
CIN III
7
2
28.6
2
0
0
Cervical cancer stage Ia
1
0
0
Cervical cancer stage IIb
2
0
0
Carcinoma in situ of the cervix
expression of E2 was found in the cancer tissue or chronic inflammation tissue (Table 1).
Discussion The coding gene sequence of the HPV18E2 protein was located in the 2817–3914 position of the HPV genome. This protein consists of 365 amino acids [7]. E2 is a transactivator involved in the transcription of viral DNA and can delay the mitosis of chromosome. In malignant tumor relevant to HPV, viral DNA often integrates in host chromosome. However, this kind of integration is rarely found in precancerous lesion or CIN. Only integrated HPV has malignant transformation capacity, because HPV-DNA controls the genetic product of the host. Therefore, integration is generally thought to be an activation mechanism of cancer from precancerous lesion. HPV 16 and 18 are two major types of cervical cancer–causing virus. A previous study has found that HPV 18 infection was more common in adenocarcinoma and adenosquamous carcinoma [8], with higher chance of pelvic lymph node metastasis and interstitial infiltration and higher recurrence rate. The integration of the HPV18 gene into the host cell is critical for cancer development. This process involves the encoding of the two proteins E1 and E2. Im et al. [9] found that E2 can bind to early promoter of HPV 18 to inhibit the transcription of E6 and E7. After a virulent form of HPV infects the cervical epithelium, the viral DNA integrates into the host chromosome and causes the continuous expression of the E6 and E7, which is the main mode by which cervical cancer develops [10]. Therefore, loss of E2 expression causes the activation of the cancer gene. In cell strains infected with HPV 18, E2 is inactivated, which indicates that the E2 inhibits the progress of cancer. Ectopic expression of the E2 in cervical cancer strains strongly inhibits the proliferation of cells. Introducing E2 into cancer cells infected with HPV can inhibit cell growth, hold the cell in the G1 phase, and promote cell aging and apoptosis. This is an intrinsic function of the E2, which is independent of the cancer-inhibiting proteins E6 and E7 [11].
In addition, the E2 induces apoptosis in HPV-negative cells. In HeLa cells, after E6 is inhibited by E2, the P53 gene is stabilized, and the P21 which is a negative regulating gene of cell cycle is activated, which holds the cell in the G1 phase. Inhibition of the E7 causes cell aging via the pRb pathway. Research has shown that E2 had highest expression at CIN stage while the E2 expression was lost in invasive cervical cancer tissue [12]. In the 30 cases included in this study, E2 expression is detected via RTPCR, and the results showed that E2 had a highest expression rate in Grade I CIN (60 %); expression rates in Grades II and III CIN, meanwhile, were 33.3 and 28.6 %, respectively. However, no expression of E2 was found in cancer tissue or chronic inflammation tissue. This is because during late-stage CIN or in highly malignant cervical cancer, E2 gene is broken during linear integration of the viral DNA into the genome. Therefore, we propose that the lesion tends to transform into malignant cancer when no E2 expression is detected during the early stage of cervical lesion. We believe that the E2 protein can be used as the target protein for novel therapeutic options. From the perspective of immune intervention, the therapeutic target proteins for late-stage CIN and highly malignant cervical cancer should be the E6 and E7, because both are highly expressed in the lesion tissue and necessary for cells to stay in conversion status. However, during early-stage infection or low malignant CIN, the E2 protein is the main component of the immune system because it is highly expressed at this stage. It can also supplement the expressions of the E6 and E7 proteins. Research has found that in cottontail rabbit papillomavirus (CRPV) model, cellular response to E2 is relevant to the regression of HPV lesion [13]. Clinical evidence has also proved that elevated level of Th cell (specific to the COOH end of the E2 protein) can be detected in women diagnosed with CIN with HPV 18 infection during the regression of lesion. This indicates that the cellular immunological responses to the E2 protein can help remove the virus. If a vaccine specific to HPV18E2 can be developed, then the lesion caused by HPV 18 will be halted at the stage of CIN and highly malignant lesion will be removed before its transformation into cancer, which will effectively lower the incidence of cervical cancer.
References 1. Bray, F., Sankila, R., Ferlay, J., et al. (2002). Estimates of cancer incidence and mortality in Europe in 1995. European Journal of Cancer, 38, 99–166. 2. Vizcaino, A. P., Moreno, V., Bosch, F. X., et al. (1998). International trends in the incidence of cervical cancer: i. Adenocarcinoma and adenosquamous cell carcinomas. International Journal of Cancer, 75, 536–545.
123
Cell Biochem Biophys 3. Ma, Y., Yu, C., Leng, Z., et al. (2004). Investigation for the relation between HPV infection and cervical squamous intraepithelial lesion and cervical carcinoma. Chongqing Medical Journal, 33, 1011–1013. 4. Resnick, R. M., Cornelissen, M. T. E., Wright, D. K., et al. (1990). Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. Journal of the National Cancer Institute, 82, 1477–1484. 5. Wang, Y., Li, L., Li, Y., et al. (2006). Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells. Acta Academiae Med Mil Tertiae, 28, 1280–1282. 6. Clifford, G. M., Smith, J. S., Aguado, T., et al. (2003). Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. British Journal of Cancer, 89, 101–105. 7. Albers, A., Abe, K., & Hunt, J. (2005). Antitumor activity of human papillomavirus type 16 E7-specific T cells against virally infected squamous cell carcinoma of the head and neck. Cancer Research, 65, 11146–11155. 8. Thierry, F., Benotmane, M. A., Demeret, C., et al. (2004). A genomic approach reveals a novel mitotic pathway in papillomavirus carcinogenesis. Cancer Research, 64, 895–903.
123
9. Im, S. S., Wilczynski, S. P., Burger, R. A., et al. (2003). Early stage cervical cancers containing human papillomavirus type 18 DNA have more nodal metastasis and deeper stromal invasion. Clinical Cancer Research, 9, 4145–4150. 10. Francis, D. A., Schmid, S. I., & Howley, P. M. (2000). Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells. Journal of Virology, 74, 2679–2686. 11. DeFilippis, R. A., Goodwin, E. C., Wu, L., et al. (2003). Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. Journal of Virology, 77, 1551–1563. 12. Demeret, C., Garcia-Carranca, A., & Thierry, F. (2003). Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein. Oncogene, 22, 168–175. 13. Blachon, S., Bellanger, S., & Demeret, C. (2005). Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis. Journal of Biological Chemistry, 280, 36088–36098.
