Oncogene database

23063

WAPAL

wings apart-like homolog (Drosophila);WAPAL;wings apart-like homolog (Drosophila)

friend of EBNA2 (Epstein-Barr virus nuclear protein 2)|friend of EBNA2 protein|wings apart-like protein homolog

10q23.2

protein-coding

Here we show that human papillomavirus (HPV) E6 and E7 oncoproteins induce hWAPL expression. In addition, small interfering RNA (siRNA) of hWAPL suppressed the growth of tumours derived from SiHa cells in nude mice. Thus, hWAPL may be one of the effective targets of uterine cervical cancer therapy.

hWAPL is a human oncogene associated with uterine cervical cancer. Here, we demonstrate that hWAPL transcription is induced by 3-methylcholanthrene (3-MC) in the cervical carcinoma-derived cell line SiHa. hWAPL transcription was analyzed with evaluation of the mRNA and heterogeneous nuclear RNA (hnRNA) levels by quantitative real time PCR analysis. Flow cytometric analysis suggested that the alteration of hWAPL mRNA levels is independent of cell cycle profile. We also found that DMSO and some components of FBS affect hWAPL transcription. Interestingly, when the aryl hydrocarbon receptor (AhR) function was inhibited by alpha-naphthoflavone (ANF), the induction of hWAPL transcription by 3-MC was greater than that in AhR-functioning normal cells. These observations suggest that there are complex mechanisms regulating the transcription of hWAPL. Furthermore, mRNA level of a mouse homolog of hWAPL in mouse uterus was induced by 3-MC injection into the abdominal cavity. Thus, some effects from 3-MC exposure on uterus may be mediated by the unscheduled overexpression of hWAPL.

AIM: To construct the prokaryotic expression plasmid of hWAPL, induce the expression of the protein and prepare polyclonal antibody. METHODS: The hWAPL cDNA was amplified by RT-PCR from HeLa cells derived RNA and then cloned to pMD18-T according to A-T. The DNA fragment was isolated and linked to prokaryotic expression vector pET28a. The recombinant protein was induced by IPTG and identified by SDS-PAGE. Then it was injected into mice to prepare polyclonal antibody. The immunogenicity and specificity of the recombinant protein were identified by ELISA and Western blot. RESULTS: The sequencing, PCR and endonucleases digestion results showed that the hWAPL fragment was correctly inserted into pMD18-T and pET28a vectors. SDS-PAGE showed 25,000 fusion hWAPL protein was expressed in BL21 cells. The titer of polyclonal antibody was 1:3 200 by indirect ELISA. The 25,000 fusion hWAPL protein was detected by SDS-PAGE and Western blot. CONCLUSION: The hWAPL protein can be expressed by prokaryotic vector pET28a. Furthermore, the expression protein can be used as immunogen to generate antibodies with specificity.

The novel human oncogene hWAPL is associated with uterine cervical cancer. The HPV16 E5 oncoprotein could induce genomic instability in normal human cells. However, the mechanism of E5 interaction with hWAPL still awaits definition. In our present studies, the eukaryotic expression plasmids, pcDNA3-hWAPL and pcDNA3-hWAPL-E5 were constructed and carried out to vaccinate mice directly. The result that indicated the polyclonal antibody titer in immunized mice sera was increased by enzyme-linked immunosorbent assay. In addition, the proliferative responses of immunized mice spleen cells showed the optical densities values in vaccinated group remarkably higher than that in the control group. In conclusion, the recombinant plasmids could induce strong humoral and cellular immune response and exhibited great potential as therapeutic targets in the treatment of cervical cancer. However, the result didn t show significant difference in group with coexpression of HPV16 E5-hWAPL and group with only hWAPL expression. Consistent with these observations, we demonstrated that HPV16 E5 was not the optimal factor to cooperate with hWAPL in gene therapy.