High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory e ect of the cell cycledependent kinase (CDK) inhibitors (CKIs) p21 WAF1/CIP1 and p27 KIP1 . To understand whether the p21 WAF1/CIP1 or p27 KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-®ve cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21 WAF1/CIP1 and p27 KIP1 . Here, we show that in a signi®cant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21 WAF1/CIP1 than for p27 KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.

High risk HPVs (human papillomaviruses) are causally involved in the development of cervical cancer. However, other factors, such as somatic genetic alterations also play a decisive role in malignant conversion of cervical keratinocytes. Mutations and chromosomal aberrations are known to in¯uence the pattern of gene expression. Therefore we used the recently developed RT ± PCR based method of di erential display of mRNAs to detect di erences in gene expression which correlate with tumorigenicity. Non-tumorigenic HPV16-immortalized human foreskin keratinocytes (HPK IA) were compared with their tumorigenic counterparts and 49 di erent genes were identi®ed which were either up-or downregulated. The identi®ed genes encode proteins of the cytoskeleton and the extracellular matrix, the nuclear splicing apparatus, transcription regulators and membrane-associated proteins. The expression pattern of all genes was also examined by RNA ± RNA in situ hybridization in biopsies of normal cervical epithelium, precancerous lesions and cancers. Two genes, C4.8 and C21.7, are of particular interest because their expression is upregulated in a subset of high grade precancerous lesions and in over 58% of cancers. These two genes may therefore be considered as putative progression markers. C4.8 is a new member of the transmembrane 4 (TM-4) protein family which includes tumor-associated antigens such as CD63 and TAPA-1, whereas C21.7 shows no signi®cant homologies to any known genes or proteins.

o 1996 Wilcy-Liss, lac.