Cytological screening for cervical cancer or its precursors using Papanicolaou's smear test (Pap test) has been highly efficient to reduce the morbidity and mortality of cervical cancer. However, evaluation of the Pap test relies on subjective diagnostic parameters and is affected by a high rate of false-positive and false-negative results. More objective diagnostic parameters to identify truly dysplastic or neoplastic cells in cervical smears as well as in cervical biopsy samples would therefore avoid insecurity for many patients and the high screening costs associated with repeated testing. Cervical dysplasia is induced by persistent infections through highrisk types of human papillomaviruses (HPVs). Outgrowth of dysplastic lesions is triggered by increasing expression of two viral oncogenes, E6 and E7, which both interact with various cell cycle-regulating proteins. Among these is the retinoblastoma gene product pRB, which is inactivated by E7. pRB inhibits transcription of the cyclin-dependent kinase inhibitor gene p16 INK4a . Increasing expression of the viral oncogenes in dysplastic cervical cells might thus be reflected by increased expression of p16 INK4a . In line with this hypothesis, we observed marked overexpression of p16 INK4a in all cervical intraepithelial neoplasm (CIN) I lesions (n ‫؍‬ 47) except those associated with low-risk HPV types (n ‫؍‬ 7), all CIN II lesions (n ‫؍‬ 32), all CIN III lesions (n ‫؍‬ 60) and 58 of 60 invasive cervical cancers. In contrast, no detectable expression of p16 INK4a was observed in normal cervical epithelium (n ‫؍‬ 42), inflammatory lesions (n ‫؍‬ 48) and low-grade cervical lesions (CIN I) associated with low-risk HPV types (n ‫؍‬ 7). Dysplastic cells could also be identified in cervical smears using a specific p16 INK4a monoclonal antibody. These data demonstrate that p16 INK4a is a specific biomarker to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin Dl triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.Progression through the mammalian cell cycle is controlled by cyclins and cyclin-dependent kinases (cdk) (1). Cyclin gene expression is tightly regulated in a phase-specific manner. Expression of cyclin Dl precedes that of cyclin E in the G1 phase of the cell cycle (2); both proteins are required and are rate-limiting for passage through G, (3)(4)(5)(6)(7). Cyclin A is first expressed at the GI/S transition; it is required for S and M phases (8-10). Cyclin A may be a component of the DNA replication machinery (11,12) and may have a role in transcriptional control during S phase (13,14). Constitutive expression of cyclin A has been associated with tumorigenesis (15, 16); inversely, abolishment of cyclin A gene expression was recently found to cause the growth arrest of adhesiondependent cells grown in suspension (17). Overexpression of cyclin Dl (18) as well as of its partner kinase cdk4 (19) is linked to tumorigenesis, and the gene MTS1, coding for p16INK4, a cellular kinase inhibitor for cdk4, is found inactivated in a large variety of human tumor cell types (20,21). We report here a regulatory link between the expression of cyclins Dl and A. Phase-specific transcription of the human cyclin A gene (22) is mediated by a binding site for the transcription factor E2F (23). Cyclin Dl can activate cyclin A transcription through this element, and this signal is antagonized by p16INK4. MATERIALS AND METHODSReporter Plasmids and Expression Vectors. cDNAs encoding human cyclin A (15), cyclin Bi (24), cyclin Dl (3), cyclin E (25), cdk4 (26), cdc2 (27), and p16'NK4 (28) were subcloned by standard techniques in the cytomegalovirus (CMV)-based expression vector pX (10). Cyclin A promoter/reporter genes were constructed as described (22). Point mutation of the E2F site was performed by PCR and verified by sequence analysis after cloning. The inducible expression vector CMV/T was constructed by inserting the simian virus 40 polyadenylylation sequence upstream of the tetracycline-controlled promoter of plasmid pUHD10-3 (29). Insertion of cDNA coding for firefly luciferase (30), cyclin Dl, and cyclin A into CMV/T yielded plasmids luc/T, cycDl/T, and cycA/T, respectively.Cell Culture and Transfection. NIH 3T3 cells and human diploid fibroblasts from foreskin were cultured and starvation synchronized as described (22). Trans...

Infection of primary B‐lymphocytes by Epstein‐Barr virus (EBV) leads to growth transformation of these B‐cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B‐cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B‐cells by mutant virus resulted in estrogen‐dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non‐proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B‐cells. These data suggest that EBV is using a common pathway for B‐cell activation bypassing the requirement for antigen, T‐cell signals and growth factors.

Cyclin E controls progression through the G 1 phase of the cell cycle in mammalian fibroblasts and potentially in many other cell types. Cyclin E is a rate-limiting activator of cdk2 kinase in late G 1 . The abundance of cyclin E is controlled by phase-specific fluctuations in the mRNA level; in mammalian fibroblasts, mRNA is not detected under conditions of serum starvation and is accumulated upon serum stimulation, with expression starting in mid-G 1 . Here, we report the cloning of the murine cyclin E promoter. We isolated a 3.8-kb genomic fragment that contains several transcriptional start sites and confers cell cycle regulation on a luciferase reporter gene. This fragment also supports transcriptional activation by adenovirus E1A, a known upstream regulator of cyclin E gene expression. An E2F binding site which is required for G 1 -specific activation of the cyclin E promoter in synchronized NIH 3T3 cells was identified in this fragment.Regulation of cell cycle progression in mammalian cells depends on the sequential activation of a series of cyclin-dependent kinases. The catalytic activity of a given cyclin-dependent kinase subunit depends on its correct posttranslational modification and on its association with the appropriate cyclin, which acts as a regulatory subunit (reviewed in reference 28). Thus, phase-specific fluctuations in the abundance of various cyclins constitute one mechanism by which the timing of cyclindependent kinase activation is controlled. It was shown that phase-specific transcriptional activation of cellular genes contributes to the control of cell cycle progression; in particular, heterodimeric transcription factor E2F/DP (referred to as E2F [17]) mediates the activation of several cellular genes at the G 1 -to-S transition (27). More recently, it was shown that E2F binding sites determine activation at the G 1 -to-S transition of the genes encoding E2F-1 (9, 11), a subunit of the E2F/DP heterodimer, and cyclin A (26), a key regulator of the G 1 -to-S transition (24, 33), indicating a regulatory link between the ability of a cell to enter S phase and the appropriate activation of E2F during G 1 .Cyclin E (13, 18) is expressed in mid-G 1 ; it associates with and activates cdk2 kinase (6,14). Conditional overexpression of cyclin E results in a decrease in the length of the G 1 interval, consistent with an acceleration of the G 1 -to-S transition (25, 31). Microinjection of anti-cyclin E antibodies during G 1 inhibits entry into S phase in normal human fibroblasts (22), and genetic evidence suggests that cyclin E controls S phase entry during early embryogenesis in Drosophila melanogaster (12). These findings strongly suggest that cyclin E and its associated kinase are centrally involved in the decision to enter S phase in most if not all eukaryotic cells. Cyclin E mRNA is absent from serum-starved normal fibroblasts and appears in mid-G 1 (13). Regulation of cyclin E gene expression appears to be involved in the transformation of mammalian cells by viral oncogenes, since a rapid ind...