1 Unit of Biomedical Research in Cancer, National Cancer Institute/Biomedical Research Institute, National Autonomous University of Mexico, Av. San Fernando No. 22, Col. Sección 16, Tlalpan, 14080 Mexico City, Mexico
2 Department of Pathology, National Cancer Institute, Mexico City, Mexico
Correspondence
Marcela Lizano-Soberón
lizano{at}servidor.unam.mx
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a previous work (Lizano et al., 1997), we suggested that the apparent exclusive association between distinct histological types of cervical cancer and different HPV types may in part be due to differences in variants infecting the genitalia. An apparent exclusive association was found between an HPV18 variant from the African branch and squamous cell carcinoma (SCC), whereas the reference isolate, as well as a European HPV18 variant, was present in cervical cancer of glandular origin with worse prognosis (adenosquamous, adenocarcinoma and small-cell carcinomas).
Few studies have been performed that addressed the functional significance of genomic or protein variations among HPV variants. It has been reported that sequence variations observed in the long control region (LCR) of a type 16 AsianAmerican isolate may be responsible for its increased transcriptional activity (Kämmer et al., 2000).
E6 is one of the viral genes expressed early during HPV infection and plays an important role in the viral life cycle, as well as in cellular immortalization and transformation (Mantovani & Banks, 2001). The transformation role of E6 is mediated by its interaction with a variety of cellular proteins. The most studied E6 target is p53. The interaction of E6 with p53 promotes the degradation of the latter via the ubiquitin pathway (Scheffner et al., 1990
; Werness et al., 1990
). It is known that the increase in p53 levels plays a critical role in the induction of genes that result in cell-cycle arrest (Di Leonardo et al., 1994
), allowing repair of damaged DNA or the activation of apoptotic pathways (Caelles et al., 1994
; Smith et al., 1995
). Therefore, cells expressing E6 maintain low levels of functional p53, altering the normal response to DNA damage and favouring accumulation of genomic mutations. On the other hand, p53 also plays an important role in apoptosis by upregulating Bax levels (Chipuk & Green, 2004
; Zhang et al., 2004
). It has been shown that low levels of p53 have anti-apoptotic activity (Lassus et al., 1996
), whereas high levels promote apoptosis (Chen et al., 1996
).
E6 mRNA from high-risk HPVs exhibits alternative splicing patterns that generate four mRNAs called E6*IE6*IV (Czegledy et al., 1994). It has been proposed that shortened E6 (E6*I) can bind to full E6, forming a complex with E6AP and competing for binding to p53 (Pim & Banks, 1999
). In this way, E6*I could probably control p53 degradation at specific moments of the infection to inhibit cellular immortalization and viral genomic integration.
A few studies have reported functional differences among HPV16 variants. E6 protein variants of HPV type 16 showed differences in their abilities to cooperate with E7 to abrogate keratinocyte differentiation and to target the degradation of p53 (Stöppler et al., 1996). Another study showed that a variant harbouring an amino acid change (LeuVal) at position 83 enhances mitogen-activated protein kinase signalling and cooperative transformation with deregulated Notch1 signalling (Chakrabarti et al., 2004
). Nevertheless, no differences in functional activities of HPV18 viral proteins from different isolates have been reported.
The aim of this work was to determine whether nucleotide changes found in the E6 oncogene from different HPV18 variants prevalent among the Mexican population affect its oncogenic potential. We suggest a mechanism that may account in part for the observed differential biological activities of naturally occurring HPV18 isolates.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolates were characterized through the genomic sequence of the LCR and E6 and E7 genes. Direct sequencing was done with a Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham Biosciences) for PCR-amplified products obtained with specific primers as shown in Table 1.
|
Cell cultures and transfections.
C-33, NIH 3T3, HaCat and MCF7 cell lines were grown in Dulbecco's modified Eagle's medium (DMEM/F12; Gibco BRL) supplemented with 8 % fetal bovine serum (FBS). Cell lines were transfected with 4 µg DNA from either vectors alone or vectors harbouring the cloned E6 variant genes. pXJ40FLAG-derived plasmids were co-transfected with 400 ng pSV2Neo plasmid for selection. Transfections were performed in 60 cm diameter dishes with Lipofectamine Plus (Gibco-BRL) according to the manufacturer's instructions. For stable-clone selection, cells were treated 24 h after transfection with G418 at 0·8 mg ml1 in DMEM/F12 for 3 weeks. After stable selection, G418-resistant cells were cloned by dilution. Integrity of E6 in the obtained clones was tested by PCR amplification and sequencing.
RNA extraction and expression analysis.
Total RNA was obtained from transfected cells or frozen biopsies by using TRIzol (Invitrogen Life Technologies) according to the manufacturer's instructions. Samples were treated with 1 U DNase I (Gibco-BRL). The amount of RNA was determined by UV spectrophotometry and quality was assessed in 2 % agarose gels. For cDNA preparation, 2 µg total RNA was reverse-transcribed with random hexamers by using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen Life Technologies). The obtained cDNA was PCR-amplified for E6-expression analysis.
In vitro degradation assay.
For in vitro degradation assays, p53 and E6, both cloned in pcDNA vector, were in vitro-translated by using a rabbit reticulocyte lysate-based coupled transcriptiontranslation system (Promega); 10 µl p53 protein, in vitro-translated in the presence of [35S]cysteine, was incubated together with 5 µl of each HPV18 E6 protein isolate. After 3 h at 30 °C, the degradation reaction was stopped by adding 100 µl sample buffer and the proteins were resolved by SDS-PAGE and visualized by fluorography.
Western blot assays.
Total cellular proteins were extracted from transfected cells harvested from a 75 cm2 plate. Cells were pelleted and disrupted with 300 µl lysis buffer [100 mM Tris (pH 8), 100 mM NaCl, 0·5 % Nonidet P-40, 1 % apoprotein, 1 mM PMSF]. Proteins were boiled in sample buffer [125 mM Tris/HCl (pH 6·8), 1 % SDS, 2 % -mercaptoethanol and 0·01 % bromophenol blue] for 5 min and then loaded onto SDS-PAGE gels (1018 %). After electrophoresis, proteins were transferred to a Hybond-C Extra nitrocellulose membrane (Amersham Biosciences) in a wet chamber for 1 h at 100 V. Membranes were then blocked with 1x TBS containing 1 % skimmed milk and 0·1 % Tween 20, washed and incubated with the corresponding antibody [E6, p53, Bax, Actine (Santa Cruz Biotechnology)]. Horseradish peroxidase-conjugated secondary antibody was used for proteinprimary-antibody complex detection. The levels of the corresponding proteins were visualized by using the ECL system (Amersham Biosciences).
Anchorage-independent growth (agar-colony assay).
For soft-agar assay, bottom layers of 0·7 % soft agar (Sigma) were prepared in 60 mm plates. Cells were harvested by trypsinization and were counted and seeded in triplicate with 15x103 cells per 60 mm dish in a 0·5 % agar top layer. The dishes were incubated at 37 °C for 2 weeks and then the number of colonies was counted. Assays were done in triplicate. Presence of 10 or more cells was the criterion taken for a formed colony.
Tumour formation in nude mice.
NIH 3T3 cells transfected with the different isolates of E6 HPV18 were trypsinized, counted, resuspended in the smallest quantity (200 µl) of DMEM/F12 and injected into nude mice (BALB/c-Nu, between 6 and 8 weeks of age) subcutaneously in the flanks. In total, 106 cells were injected per mouse. Tumour size was measured every 7 days. After 1 month, the mice were sacrificed and the tumours were taken out, formalin-fixed and paraffin-embedded. Tumour volume was calculated by using the formula Vt=(2ab)
/6, where Vt is tumour volume, a is the largest diameter and b is the smallest diameter.
Site-directed mutagenesis assay.
Site-directed mutagenesis was performed in the E6 reference clone by using a Transformer site-directed mutagenesis kit (Clontech) in order to mutate in the E6 reference isolate each of the variations contained in the E6 African variant. The positions analysed are shown, with the intended nucleotide exchange underlined at the centre of each primer used: T251C (5'-GAATTTGCATTCAAAGATTTATTTG-3'); G266A (5'-GATTTATTTGTAGTGTATAGAG-3'); T317C (5'-GTATAGATTTCTATTCTAGAATTAG-3'); C342T (5'-GAATTAAGATATTATTCAGAC-3'); G374A (5'-GAGACACATTAGAAAAACTAAC-3'); C491A (5'-GATTTCACAAAATAGCTGGGC-3'); and A458G (5'-CGACAGGAGCGACTCCAACGAC-3'). The selected clones with the E6 ORFs were sequenced to confirm the presence of the mutated nucleotide. RNA extracted from cells transfected with E6 mutants was submitted to RT-PCR to analyse E6 expression. p53-protein levels of such clones were examined by Western blot.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
E6 transcriptional patterns in cell lines and cervical tumours
To evaluate whether nucleotide changes in E6 could have an effect on its expression, transcriptional patterns were analysed. pcDNA plasmids harbouring reference, European or African E6 oncogenes were transfected into different cell lines. After selection, G418-resistant clones were assessed for the presence of E6 by PCR and transcriptional patterns were analysed through RT-PCR (Fig. 1a, b). Evident differences were found at the transcriptional level between the African and the other two isolates, in particular the relative levels of E6 and E6*I transcripts, suggesting differences at the RNA-processing level. Fig. 1(a)
shows the presence of the two transcripts in all clones: that of 477 nt corresponds to the full-length E6 transcript and that of 295 nt to the E6*I transcript. Sequencing the corresponding PCR products confirmed the nature of the observed RT-PCR products. The most important finding in this assay was the different proportion of each transcript within the different E6 isolates. In the clones with the AsianAmerindian and the European E6, we detected higher levels of full-length E6 transcript in relation to E6*I. In contrast, expression of E6*I was evidently favoured in clones with the African E6, where levels of the full-length E6 transcript were either greatly reduced or minimal.
|
In order to define the E6 transcriptional patterns in RNA obtained from cervical tumours positive for different HPV18 isolates and to compare these patterns with results obtained in the transfected stable clones, RT-PCR was performed in material obtained from four tumour samples containing the reference E6, six samples with the European variant and three samples with the African isolate. Fig. 1(c) shows that E6 expression in tumours harbouring the African isolate is similar to that obtained in cell clones, with a clear higher proportion of E6*I than the full-length E6 transcript. Tumours harbouring reference E6 (AsianAmerindian) have higher expression of the full-length E6 transcript, whereas tumours with the European isolate show a mixed pattern, including cases with a higher proportion of the E6*I transcript (data not shown), but the majority (four out of six) presented a higher proportion of the full-length E6 transcript.
E6-protein levels in transfected cells
When analysing E6-protein expression in transfected cells, as shown in Fig. 2(a), we noticed a correlation between the previously observed E6 transcriptional patterns and E6 protein-expression levels. A clear reduction of E6 levels was seen in clones with African E6, whereas no difference was found in E6-protein levels between the reference or European clones. Nevertheless, under our conditions, we were unable to detect the E6*I protein in any case.
|
Effect on cellular proteins of differential expression of E6 within HPV18 variants
The transforming properties of E6 result from its ability to form complexes with and modulate the action of an important array of cellular proteins, some of which are involved in cell growth and differentiation. As p53 is nowadays the most-studied E6 cellular target, we were interested in evaluating whether changes in transcriptional and translational patterns of African E6 could be reflected in differences in p53 steady-state levels. As expected, cells transfected with reference or European E6 clones showed a clear reduction in p53 levels, in comparison to cells transfected with the vector alone (Fig. 2b). Interestingly, clones with African E6 showed higher levels of p53, similar to clones harbouring the vector alone. This effect may be due to the reduction of E6-protein levels found in clones harbouring this variant.
It is feasible that steady-state levels of proteins responding to p53 could also be affected in the transfected clones. Therefore, we analysed the levels of Bax protein, a pro-apoptotic member of the Bcl2 family known to be increased in response to p53 levels. Fig. 2(c) shows that, indeed, steady-state levels of Bax protein show a relationship with those of p53. Clones harbouring the reference E6 (AsianAmerindian) have clearly lower levels of Bax than clones with the vector alone or African E6. This suggests that cells harbouring this E6 might maintain higher levels of p53 and proteins regulated by it, affecting its transforming ability.
In vitro degradation of p53 is not affected by variations in E6
Although the changes already found at the transcriptional and translational levels of African E6 appear to have important biological consequences, it remained to be determined whether the two amino acid changes found in this protein could affect its ability to promote in vitro degradation of p53 via the ubiquitin pathway. To achieve this goal, we used an in vitro protein-translation system with reticulocyte lysate, where no splicing of E6 is expected and the proteolysisubiquitination pathway remains active. No apparent differences were observed in levels of in vitro-translated p53 in the presence of AsianAmerindian or African E6 proteins (Fig. 3). Therefore, it appears that the two amino acid changes between these E6 proteins do not affect their ability to promote p53 degradation in vitro. This suggests that, although very low levels of the E6 protein are present in clones containing African E6, it may maintain the same, although highly reduced, functional activity as the reference clone in promoting p53 degradation.
|
|
A conserved site among high-risk HPVs favours E6 transcript processing
E6 from type 18 is translated from a single bicistronic E6E7 mRNA, but alternatively spliced mRNAs are also frequently formed, generating E6*IE6*IV mRNAs that potentially encode proteins with the same N-terminal region as seen in the full-length E6 protein (Czegledy et al., 1994). Nevertheless, the mechanisms involved in such splicing are not well-known; our results show that clones harbouring African E6 present a higher proportion of the E6*I transcript, in relation to the full-length E6. Therefore, we proceeded to determine which nucleotide changes in E6 could be implicated in altering such transcriptional patterns. We introduced every change present in African E6 into the reference clone, except for those changes shared with the European E6. As shown in Fig. 4
(a), replacement of nt 491 (CA) changes the E6 transcriptional pattern obtained with the reference clone, generating a pattern similar to that of the African E6, with a higher proportion of E6*I than full-length E6. It is worth mentioning that nt 491 corresponds to aa 129, a position that is highly conserved within oncogenic HPV types (Pim et al., 1994
). Meanwhile, no other change produced such an effect, including that at position G374A, which is located near the spliceosome branch point (Brow, 2002
). When comparing p53 steady-state levels between cell clones with E6 mutants (C491A and G374A), we confirmed the correlation between E6-expression patterns and p53-protein levels (Fig. 4b
). Cells with reference E6 mutated at position 491 (CA) have similar p53 levels to cells with African E6, whereas cells with the G374A mutant showed no change in E6 transcriptional pattern and, consequently, maintained the same p53-protein levels as cells with the reference E6.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hecht et al. (1995) suggested that genomic variability of different HPV18 isolates might be responsible for the wide spectrum of pathologies associated with this viral type. These authors identified an HPV18 variant absent in cervical cancer, but present in 40 % of intraepithelial lesions, suggesting a lower oncogenic potential.
We reported previously the identification of three different HPV18 isolates in the Mexican population (Lizano et al., 1997). Our findings showed an apparent exclusive association between an African isolate and SCC, in contrast to the reference clone, which was found to be associated with other histological types of cervical cancer with worse prognoses. This finding suggested a different biological behaviour for this variant; this is supported by the recent results of De Boer et al. (2005)
, who identified a similar HPV18 variant belonging to the African branch exclusively in SCCs that, although sharing some of the changes with our African isolate, differs in the E7 and LCR sequences. It is worth mentioning that all samples with the African variant previously identified by Lizano et al. (1997)
had the same conserved changes in E6, E7 and the LCR.
We found that our European variant is similar in the LCR fragment to a European variant reported previously (Ong et al., 1993). Although our African variant is not identical to any known LCR or E6 sequences (De Boer et al., 2005
), it has great resemblance to the African branch reported by Ong et al. (1993)
within the LCR. Interesting differences were found between the African LCR and the reference clone. A 7 bp deletion (positions 72457251) and mutations found at the enhancer region, which is known to harbour consensus sequences for transcriptional factors (AP1, TEF1, OCT1), suggest the likelihood of altered transcriptional functions. Therefore, we are already testing this possibility by determining transcriptional activities of the different HPV18 isolates.
As expected for E7, one of the most conserved genes among HPVs, few changes were found at the nucleotide level and only one amino acid change for the African variant E7 (nt 864, leading to N92S), which does not discard the possibility of an altered E7 function. It is worth mentioning that specific viral sequence variations may lead to altered biological functions of the translated proteins, which might affect the clinical outcome of the infection. The two amino acid changes predicted for the African E6 protein (positions 80 and 129) might suggest changes in its oncogenic function, as these positions are highly conserved within oncogenic HPV types (Pim et al., 1994). Nevertheless, in vitro degradation analysis of p53 showed that these changes in E6 do not affect its ability to promote p53 degradation. Although this is one of the most important interactions of E6, it should not leave aside the existence of multiple targets of E6 that might also be affected. It is important to consider that mutations at the amino acid level might affect the protein's ability to interact with cellular proteins and promote transformation; nevertheless, silent changes are no less important, as they could also affect gene expression at the transcriptional level.
When analysing E6 expression, we found that transcriptional patterns were visibly different within HPV18 variants. A higher proportion of the E6*I transcript in relation to the full-length E6 was found exclusively in cells transfected with the African E6, in clear contrast to that observed with the reference (AsianAmerindian) or European E6, where full-length E6 transcripts were found in a higher proportion than the E6*I transcripts. This finding suggests that nucleotide changes present in African E6 may promote its alternative splicing, reducing functional E6-protein levels. These data are supported by results of other authors who found that E2 is downregulated by alternative splicing in tumours harbouring an European HPV16 variant (Ordóñez et al., 2004). Although the proportion of the E6*I transcript is highly augmented with African E6, we were unable to detect the corresponding protein, which may be related to its intrinsic instability (Pim et al., 1997
).
Despite the fact that our analysis with tumours was done with a limited number of cases, we always noticed that similar E6 transcriptional patterns were present in tumours harbouring the African and AsianAmerindian isolates, suggesting a possible correlation between E6 transcriptional patterns found in tumours and transfected cell lines. However, even though tumours harbouring the European isolate exhibited a mixed pattern of E6 transcripts, the majority of them (four out of six tumours) showed a high proportion of the full-length E6 transcripts. Our results should be enhanced by the analysis of a greater number of tumours.
In relation to HPV mRNA processing, although it has been shown that changes at donor or acceptor sites affect transcript generation, there is no evidence for the involvement of other sites in this process. Our results obtained through site-directed mutagenesis showed the importance of position 491 in E6 mRNA processing, as reversion of this position in the reference clone changed the E6 transcriptional pattern to that observed in cells transfected with the African E6. As this position is not the donor or acceptor site or the branch point, it should be determined whether mutation of this site may affect the binding of cellular factors involved in formation of the spliceosome.
In this study, we observed that the low levels of full-length E6 transcript in cells transfected with the African E6 led to a reduction in E6-protein levels, a fact that was reflected strongly in p53-protein levels. As expected, cells with the reference E6 showed a reduction in p53 levels, whereas cells with African E6 showed levels of p53 similar to that in cells without E6. We also observed that steady-state levels of the Bax protein correlated with p53 levels observed in different cellular clones. Higher levels of Bax found in cells transfected with the African E6 may alter cell receptivity to apoptotic stimuli. Mutation of reference E6 (C491A) reversed the pattern of E6 transcripts to that of the African E6 and, consequently, exhibited a clear increase in p53 levels. This could explain, in part, the less aggressive behaviour proposed for the African variant.
In order to determine possible functional consequences of our results, we analysed the tumour-forming potential of different HPV type 18 E6 isolates in nude mice. All cells transfected with the different isolates could form tumours sooner or later; the growth rate was clearly slower for tumours with the African E6. These results support the hypothesis that intratype changes in E6 could delay transformation processes of a cell containing the African isolate. This could explain in part a diminished oncogenic potential, as well as a better prognosis, for tumours harbouring African isolates in contrast to other isolates of HPV18.
Sequence variation within one or more viral proteins may lead to altered biological functions and affect the clinical outcome of infection. Multiple mechanisms are involved in cell transformation. Reduction of one or more viral transforming elements does not assure loss of the virus's transforming ability, but may drive differences in the expected biological behaviour, delaying the transformation process.
Although our findings of different proportions of E6 transcripts within HPV18 variants seem to support the observed differences in their biological behaviour, it should be stressed that differences in E6 transcript processing could affect E7 translation, in spite of the fact that previous reports show that E7 is translated efficiently both from the bicistronic E6E7 mRNA and from its spliced transcript (Stacey et al., 1995). Nevertheless, it remains important to dissect the role of the other viral genes, such as E2, E5 and mainly E7, in modulating the oncogenic potential of different HPV18 variants, as the combined function of different viral genes is critical in cell transformation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berumen, J., Ordoñez, R. M., Lazcano, E. & 7 other authors (2001). Asian-American variants of human papillomavirus 16 and risk for cervical cancer: a casecontrol study. J Natl Cancer Inst 93, 13251330.
Bosch, F. X., Lorincz, A., Muñoz, N., Meijer, C. J. L. M. & Shah, K. V. (2002). The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 55, 244265.
Brow, D. A. (2002). Allosteric cascade of spliceosome activation. Annu Rev Genet 36, 333360.[CrossRef][Medline]
Burger, R. A., Monk, B. J., Kurosaki, T., Anton-Culver, H., Vasilev, S. A., Berman, M. L. & Wilczynski, S. P. (1996). Human papillomavirus type 18: association with poor prognosis in early stage cervical cancer. J Natl Cancer Inst 88, 13611368.
Caelles, C., Helmberg, A. & Karin, M. (1994). p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 370, 220223.[CrossRef][Medline]
Chakrabarti, O., Veeraraghavalu, K., Tergaonkar, V., Liu, Y., Androphy, E. J., Stanley, M. A. & Krishna, S. (2004). Human papillomavirus type 16 E6 amino acid 83 variants enhance E6-mediated MAPK signaling and differentially regulate tumorigenesis by Notch signaling and oncogenic Ras. J Virol 78, 59345945.
Chen, X., Ko, L. J., Jayaraman, L. & Prives, C. (1996). p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev 10, 24382451.[Abstract]
Chipuk, J. E. & Green, D. R. (2004). Cytoplasmic p53: Bax and forward. Cell Cycle 3, 429431.[Medline]
Czegledy, J., Evander, M., Hernadi, Z., Gergely, L. & Wadell, G. (1994). Human papillomavirus type 18 E6* mRNA in primary tumors and pelvic lymph nodes of Hungarian patients with squamous cervical cancer. Int J Cancer 56, 182186.[Medline]
Dalstein, V., Riethmuller, D., Prétet, J.-L., Le Bail Carval, K., Sautière, J.-L., Carbillet, J.-P., Kantelip, B., Schaal, J.-P. & Mougin, C. (2003). Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal French cohort study. Int J Cancer 106, 396403.[CrossRef][Medline]
De Boer, M. A., Peters, L. A. W., Aziz, M. F., Siregar, B., Cornain, S., Vrede, M. A., Jordanova, E. S. & Fleuren, G. J. (2005). Human papillomavirus type 18 variants: histopathology and E6/E7 polymorphisms in three countries. Int J Cancer 114, 422425.[CrossRef][Medline]
Di Leonardo, A., Linke, S. P., Clarkin, K. & Wahl, G. M. (1994). DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev 8, 25402551.[Abstract]
Halpern, A., McBride, A., Myers, G., Baker, C., Wheeler, C. M. & Doorbar, J. (editor) (1997). Human Papillomaviruses. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos, NM: Los Alamos National Laboratory.
Hecht, J. L., Kadish, A. S., Jiang, G. & Burk, R. D. (1995). Genetic characterization of the human papillomavirus (HPV) 18 E2 gene in clinical specimens suggests the presence of a subtype with decreased oncogenic potential. Int J Cancer 60, 369376.[Medline]
Hildesheim, A., Schiffman, M., Bromley, C. & 12 other authors (2001). Human papillomavirus type 16 variants and risk of cervical cancer. J Natl Cancer Inst 93, 315318.
Im, S. S., Wilczynski, S. P., Burger, R. A. & Monk, B. J. (2003). Early stage cervical cancers containing human papillomavirus type 18 DNA have more nodal metastasis and deeper stromal invasion. Clin Cancer Res 9, 41454150.
Kämmer, C., Warthorst, U., Torrez-Martinez, N., Wheeler, C. M. & Pfister, H. (2000). Sequence analysis of the long control region of human papillomavirus type 16 variants and functional consequences for P97 promoter activity. J Gen Virol 81, 19751981.
Lassus, P., Ferlin, M., Piette, J. & Hibner, U. (1996). Anti-apoptotic activity of low levels of wild-type p53. EMBO J 15, 45664573.[Abstract]
Lizano, M., Berumen, J., Guido, M. C., Casas, L. & Garcia-Carranca, A. (1997). Association between human papillomavirus type 18 variants and histopathology of cervical cancer. J Natl Cancer Inst 89, 12271231.
Mantovani, F. & Banks, L. (2001). The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 20, 78747887.[CrossRef][Medline]
McLachlin, C. M., Tate, J. E., Zitz, J. C., Sheets, E. E. & Crum, C. P. (1994). Human papillomavirus type 18 and intraepithelial lesions of the cervix. Am J Pathol 144, 141147.[Abstract]
Moscicki, A.-B., Hills, N., Shiboski, S. & 9 other authors (2001). Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females. JAMA 285, 29953002.
Myers, G., Bernard, H. U., Delius, H., Favre, J., Icenogle, J., Van Ranst, M. & Wheeler, C. M. (editors) (1994). Human Papillomaviruses. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos, NM: Los Alamos National Laboratory.
Ong, C.-K., Chan, S.-Y., Saveria Campo, M. & 8 other authors (1993). Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups. J Virol 67, 64246431.[Abstract]
Ordóñez, R. M., Espinosa, A. M., Sánchez-González, D. J., Armendáriz-Borunda, J. & Berumen, J. (2004). Enhanced oncogenicity of Asian-American human papillomavirus 16 is associated with impaired E2 repression of E6/E7 oncogene transcription. J Gen Virol 85, 14331444.
Pim, D. & Banks, L. (1999). HPV-18 E6*I protein modulates the E6-directed degradation of p53 by binding to full-length HPV-18 E6. Oncogene 18, 74037408.[CrossRef][Medline]
Pim, D., Storey, A., Thomas, M., Massimi, P. & Banks, L. (1994). Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo and immortalisation of primary BMK cells. Oncogene 9, 18691876.[Medline]
Pim, D., Massimi, P. & Banks, L. (1997). Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth. Oncogene 15, 257264.[CrossRef][Medline]
Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 11291136.[CrossRef][Medline]
Schlecht, N. F., Kulaga, S., Robitaille, J. & 8 other authors (2001). Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. JAMA 286, 31063114.
Smith, M. L., Chen, I. T., Zhan, Q., O'Connor, P. M. & Fornace, A. J., Jr (1995). Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene 10, 10531059.[Medline]
Stacey, S. N., Jordan, D., Snijders, P. J. F., Mackett, M., Walboomers, J. M. M. & Arrand, J. R. (1995). Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame. J Virol 69, 70237031.[Abstract]
Stöppler, M. C., Ching, K., Stöppler, H., Clancy, K., Schlegel, R. & Icenogle, J. (1996). Natural variants of the human papillomavirus type 16 E6 protein differ in their abilities to alter keratinocyte differentiation and to induce p53 degradation. J Virol 70, 69876993.[Abstract]
Walboomers, J. M. M., Jacobs, M. V., Manos, M. M. & 7 other authors (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189, 1219.[CrossRef][Medline]
Werness, B. A., Levine, A. J. & Howley, P. M. (1990). Association of human papillomavirus type 16 and 18 E6 proteins with p53. Science 248, 7679.[Medline]
WHO (2004). The World Helath Report 2004: Changing History, Statistical Annex. Geneva: World Health Organization (http://whqlibdoc.who.int/whr/2004/924156265X.pdf).
Xi, L. F., Koutsky, L. A., Galloway, D. A., Kuypers, J., Hughes, J. P., Wheeler, C. M., Holmes, K. K. & Kiviat, N. B. (1997). Genomic variation of human papillomavirus type 16 and risk for high grade cervical intraepithelial neoplasia. J Natl Cancer Inst 89, 796802.
Zehbe, I., Wilander, E., Delius, H. & Tommasino, M. (1998). Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype. Cancer Res 58, 829833.[Abstract]
Zhang, H.-G., Wang, J., Yang, X., Hsu, H.-C. & Mountz, J. D. (2004). Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene 23, 20092015.[CrossRef][Medline]
zur Hausen, H. (2002). Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2, 342350.[CrossRef][Medline]
Received 3 February 2005;
accepted 7 June 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |