Human Papillomavirus Type 16 Mutant E7 Protein Induces Oncogenic Transformation via Up-regulation of Cyclin A and cdc25A *
A new mutant human papillomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China). A previous study showed that the HPV16 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WE7, was concentrated in the nucleus. With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay. The effect of HPV16 HBE7 on cell cycle was examined by flow cytometry. Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPV16 HBE7 protein on the expression of regulation factors associated with G1/S checkpoint. The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity. NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A. These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.
cervical cancer cells, and is vital to maintain the malignant phenotype of these cells (10) . Moreover, HPV16 E7 can transform a variety of cell lines, including NIH/3T3 mouse fibroblasts (27) . The most well-characterized biochemical property of HPV16 E7 is its ability to bind to the unphosphorylated retinoblastoma tumor suppressor protein (Rb) and disrupt Rb-E2F complexes, resulting in a loss of the G1/S checkpoint (1, 7, 35, 38) . The HPV16 E7 protein is a 98-amino-acid nuclear phosphoprotein and can be divided into three main domains -CR1, aa 1-15; CR2, aa 16-37; and CR3, aa 38-98. All 3 domains are essential for the manifestation of the biological properties of E7 (33) . CR1 and CR2 mediate binding of the viral oncoproteins to an overlapping set of cellular proteins, including the product of the retinoblastoma gene (pRB), cyclin A and cyclin E (18, 24, 36) . The CR3 domain contains two "CXXC" sequences participating in Zn binding (9, 23) . It was reported that zinc finger structure at the C-terminal was essential for HPV16 E7 to stabilize its structure and biological function (30) .
Previous studies showed variants of HPV16 E7 gene were found in widely separated locations (5, 26, 31) . A mutant E7 (HPV16 HBE7) protein derived from a clinical HPV16 was isolated from Hubei province in China (16) . It was found that there were two mutations in HPV16 HBE7 ( GenBank accession no. AF393782). What is more, a non sense mutation was found in codon 43 which made HPV16 HBE7 into a truncated protein, corresponding to the first 43 amino acids of HPV16 WE7. Generally speaking, the great variance of gene structure in the virus will lead to changes of its immunological characteristics and its biological functions, for example, mutations leading to changes in the structure of coronaviruses produces changes in host range and virulence (27) . Previous research indicated that the HBE7 protein could induce mice to produce humoral immunity identical with the WE7 protein but with weaker specific cellular immunity (16) . The integrity of the C-terminal of the truncated HBE7 protein affects its intracellular localization (17) and may affect its transformation activity. This research shows that HPV16 HBE7 (the HPV16 E7 fragment), which is predominantly cytoplasmic, is also competent for transformation of NIH/3T3 cells and can efficiently activate expression of cyclin A and cdc25A.
NIH/3T3 cells were purchased from the Cancer Research Institute of Zhongnan University (Hunan, China) and cultured in DMEM supplemented with 10% BCS at 37 ℃ in 5% CO 2 atmosphere.
Recombinants of pcDNA3.1-HPV16HBE7 and pcDNA3.1-HPV16WE7 expressing HBE7 or WE7 respectively were generated previously (37) . To evaluate the transformation ability of HPV16 HBE7 and its potential to regulate the G1/S phase of cell cycle, NIH/3T3 cells (1.5 ×10 5 /well) were transfected with 0.8 µg plasmid DNA using lipofectamine TM 2000.
Stable clones were selected using G418 (Wuhan TianYuan BioTechnologies co, China) at a concentration of 600µg/mL. Empty vector pcDNA3.1 (-) was also stably transfected into NIH/3T3 cells as a control.
Expression of HPV16 E7 protein in stable clones was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs of NIH/3T3 cells were extracted by Trizol reagent (MBI Fer-mentas). 5µg of total RNA was reverse-transcribed to cDNA with a first-strand cDNA synthesis kit (MBI Fermentas). The PCR was performed in a 50µL volume under the following conditions: initial denaturation step of 94 ℃ for 5 min, cycling step of denaturation at 94 ℃ for 60 s, annealing for 60s and extension at 72 ℃ for 60s. The mRNA levels of β-actin served as an internal control. The products of PCR were identified by agarose gel analysis.
Changes of cell morphology in stable NIH/3T3 cell transfectants with pcDNA3.1-HPV16HBE7 were observed by transmission electron microscope (Olympus, Japan). In the process of culturing, the samples of 5 ×10 6 cells were collected and fixed with 2.0% glutaraldehyde, dehydrated, embedded, sectioned and finally visualized via transmission electron microscope.
The cell morphology of parental NIH/3T3 cells was set as a control.
The growth rate of NIH/3T3 cells stably expressing HBE7 in vitro was measured using the 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method (25) . Briefly, cells were seeded into 96-well plates (2×10 3 cells/well). On the day of harvest, 100μL of spent medium was replaced with an equal volume of fresh medium containing 10% MTT 5 mg/mL stock. Plates were incubated at 37 ℃ in a 5% CO 2 incubator for 4 h, then 100μL of dimethyl sulphoxide (DMSO) was added to each well and plates were shaken at room temperature for 10 min.
Soft agarose assay was essentially performed according to previous methods (22) . The NIH/3T3 cells transfected with pcDNA3.1-HPV16HBE7 and parental NIH/3T3 cells were respectively suspended in 0.35% agarose with complete DMEM medium, plated at a density of 1×10 4 cells per well, previously coated with 0.7% agarose and maintained at 37˚C in 5% CO 2 atmosphere. The cultures were maintained for 3 weeks and colonies>150µm in diameter were counted under an inverted microscope at the magnification of 10 times.
The distribution of cells in the cell cycle phases was determined by FACS analysis of the DNA content.
The cyclinA promoter activity was determined by dual-luciferase reporter assay. NIH/3T3 cells were grown in 24 well plates to about 70%-80% confluence and were cotransfected with recombinants of pcDNA3.1-
RT-PCR was performed in a semiquantitative manner with cyclinA, cdc25A, cyclin E and cdk2.
Primers for amplification of each gene are listed in
Data were presented as mean ± SD and analyzed with SPSS 11.0 software. P < 0.05 was considered statistically significant.
A specific band could be seen in both NIH/3T3 cells stably transfected with pcDNA3.1-HPV16HBE7 and NIH/3T3 cells stably transfected with pcDNA3.1-HPV16WE7 (Fig.1) . It showed that NIH/3T3 cells stably expressing HBE7 (NIH/3T3-HBE7) and NIH/ 3T3 cells stably expressing WE7 (NIH/3T3-WE7)
were successfully obtained.
Transmission electron microscopy revealed that NIH/3T3 cells stably expressing HBE7 (NIH/3T3-HBE7) grew quite well. There were several bigger and abnormally shaped nucleoli in NIH/3T3-HBE7 cells and more pathologic nuclear phase and immature cell organs were found in NIH/3T3-HBE7 cells than in parental NIH/3T3 cells (Fig. 2) . (Fig. 3) .
The tumorigenic potential of transformed cells was determined from their ability to grow as anchorageindependent colonies in soft agarose. Small colony formation could be seen in NIH/3T3-HBE7 cells two weeks later. At 15 to 21 days, obvious cell colonies were formed. On day 21, NIH/3T3-HBE7 cells could form some colonies in the soft agarose, while parental NIH/3T3 cells could not form colonies (Fig. 4) . These data suggested there was tumorigenesis in the HBE7 protein.
The effect of the HBE7 gene on cell cycle distribution was determined to gain insights into the mechanism of its proliferative activity. As can be seen in Table 2 , the presence of the HBE7 gene resulted in The results showed that compared with pcDNA3.1(-)
transfecting the cells, the activity of cyclin A promoter increased by 2 times in HBE7 transfecting cells (Fig. 5 ). These results demonstrate that HPV16HBE7 protein promotes the proliferation of NIH/3T3 cells and the HBE7 gene is a transforming gene.
To further investigate the potential of HBE7 to regulate the G1/S phase of cell cycle, the expression level of regulation factors associated with G1/S checkpoint was examined. Semi-quantitative RT-PCR results are presented in Fig.6 . The relative mRNA expression levels of each gene are listed in Table 3 .