(HPV)-activating cellular transcription factor Brn-3a ... - Clinical Science

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intraepithelial neoplasia stage 3). Daniel NDISANG*, Vishwanie BUDHRAM-MAHADEO*, Albert SINGER† and David S. LATCHMAN*. *Institute of Child Health, ...
Clinical Science (2000) 98, 601–602 (Printed in Great Britain)



C O R R E S P O N D E N C E

Widespread elevated expression of the human papilloma virus (HPV)-activating cellular transcription factor Brn-3a in the cervix of women with CIN3 (cervical intraepithelial neoplasia stage 3) Daniel NDISANG*, Vishwanie BUDHRAM-MAHADEO*, Albert SINGER† and David S. LATCHMAN* *Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K., and †Department of Women’s and Children’s Health, The Whittington Hospital, Highgate Hill, London N19 5NF, U.K. It is now well established that the human papilloma virus (HPV) types 16 and 18 encode transforming proteins (E6 and E7) and play a key role in human cervical cancer [1]. However, both HPV-16 and HPV-18 can be detected in women with undetectable or minimal cervical abnormality [2], suggesting that cellular factors may regulate the progression of HPV-induced transformation. Interestingly, it has been shown that human individuals with a p53 tumour suppression protein having an arginine rather than a proline residue at position 72 show an enhanced risk of cervical cancer [3]. This correlates with the more efficient degradation of the arginine-containing form of the protein by the HPV E6 protein, indicating that cellular factors modulating HPV protein function can alter the risk of transformation. A number of cellular transcription factors have been shown to regulate the expression of the genes encoding E6 and E7. Evidently, therefore, alterations in such factors could also affect the risk of cervical cancer by altering the expression of E6\E7 rather than their activity. In particular, we have demonstrated that the cellular POU family transcription factor Brn-3a can strongly activate the upstream regulatory region of the virus genome which controls the expression of the genes encoding E6 and E7 [4]. Moreover, inhibition of Brn-3a expression in cervical cells expressing HPV dramatically reduces HPV gene expression and inhibits cellular transformation, as assayed by saturation density and anchorage-independent growth in vitro and the ability to form tumours in nude mice in vivo [5]. These effects are not observed when Brn-3a expression is reduced in a cervical cell line lacking HPV genomes.

Most importantly, we have demonstrated that Brn-3a is overexpressed approx. 300-fold in the transformation zone of women with CIN3 (cervical intraepithelial neoplasia stage 3) compared with the level observed in women with no or minimal cervical abnormality [6]. Hence, elevated expression of Brn-3a in the cervix of women with CIN3 is likely to play a critical role in activating HPV gene expression and, therefore, in cervical transformation. The elevation of Brn-3a levels could be a localized effect confined to the area of the lesion or, alternatively, might represent a more widespread elevation, which could act as a risk factor for cervical transformation in cells containing HPV DNA. To distinguish between these possibilities, we measured Brn-3a expression in cervical biopsies from women with CIN3, which were taken at successive 5 mm intervals from the lesion site. None of the women was menstruating at the time of biopsy. Brn-3a expression was measured at the mRNA level using a reverse transcriptase\PCR assay as described in our previous study [6], and was compared with the level in cervical samples from a number of women without detectable cervical abnormality. In these studies (Figure 1), the high level of expression of Brn-3a found in the CIN3 lesion was maintained right across the normal areas of the cervix. Thus normal cervical areas from women with CIN3 showed a greatly elevated Brn-3a level compared with that found in samples of normal cervix from 19 control subjects without detectable cervical abnormality. This indicates that the increase in Brn-3a levels is not confined to the CIN3 lesion. Rather, women with CIN3 exhibit grossly elevated levels of Brn-3a throughout the cervix. It is likely, therefore, that

Key words : Brn-3a, cervical cancer, human papilloma virus, transforming proteins. Abbreviations : CIN3, cervical intraepithelial neoplasia stage 3 ; HPV, human papilloma virus. Correspondence : Professor David S. Latchman (e-mail d.latchman!ich.ucl.ac.uk).

# 2000 The Biochemical Society and the Medical Research Society

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Figure 1

Elevated expression of Brn-3a throughout the cervix of women with CIN3

Brn-3a mRNA levels in the cervix of four women with CIN3 are compared with the average level in normal individuals. In the case of the women with CIN3, the mRNA level is shown for cervical biopsies taken from the CIN3 region itself and at 5 mm intervals away from it. Note the greatly elevated level of Brn-3a mRNA throughout the cervix of women with CIN3 compared with the level in 19 normal individuals without detectable cervical abnormality. genetic or environmental differences between different individuals result in some women having much higher levels of Brn-3a than others. Following HPV infection, such elevated levels of Brn-3a would induce transcription of the HPV genome and cellular transformation in the area at the junction of the ecto- and endo-cervix where HPV DNA is located and where tumours arise. Hence, elevated Brn-3a levels are likely to represent a significant risk factor, enhancing the likelihood of rapid disease progression following HPV infection in women with this elevation.

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REFERENCES 6 1 Durst, M., Gissmann, L., Ikenberg, H. and Zur Hausen, H. (1983) A papilloma virus DNA from a cervical carcinoma and its prevalence in cancer biopsy

samples from different geographic regions. Proc. Natl. Acad. Sci. U.S.A. 80, 3812–3815 Cox, M. F., Meanwell, C. A., Maitland, N. J., Blackledge, G., Scully, C. and Jordan, J. A. (1986) Human papillomavirus type-16 homologous DNA in normal human ectocervix. Lancet ii, 157–158 Storey, A., Thomas, M., Kalita, A. et al. (1998) Role of a p53 polymorphism in the development of human papillomavirus. Nature (London) 393, 229–234 Morris, P. J., Theil, T., Ring, C. J. A., Lillycrop, K. A., Mo$ ro$ y, T. and Latchman, D. S. (1994) The opposite and antagonistic effects of the closely related POU family transcription factors on the activity of a target promoter are dependent upon differences in the POU domain. Mol. Cell. Biol. 14, 6907–6914 Ndisang, D., Budhram-Mahadeo, V., Morris, P. J. and Latchman, D. S. (1999) The Brn-3a transcription factor plays a critical role in regulating HPV gene expression and determining the growth characteristics of cervical cancer cells. J. Biol. Chem. 274, 28521–28527 Ndisang, D., Morris, P. J., Chapman, C., Ho, L., Singer, A. and Latchman, D. S. (1998) The HPV activating transcription factor Brn-3a is over expressed in CIN3 lesions. J. Clin. Invest. 101, 1687–1692 Received 22 November 1999

# 2000 The Biochemical Society and the Medical Research Society