Concordant Induction of Cyclin E and p21 cip1 in Differentiated Keratinocytes by the Human Papillomavirus E7 Protein Inhibits Cellular and Viral DNA Synthesis 1 Yichun Jian, Brian A. Van Tine, Wei-Ming Chien, George M. Shaw, Thomas R. Broker, and Louise T. Chow 2 Departments of Biochemistry and Molecular Genetics [Y. J., W-M. C., T. R. B., L. T. C.], Pathology [B. A. V. T.], and Medicine [B. A. V. T., G. M. S.], The Howard Hughes Medical Institute [G. M. S.], University of Alabama at Birmingham, Birmingham, Alabama 35294-0005 Abstract Productive infections by human papillomaviruses (HPVs) occur only in differentiated keratinocytes in squamous epithelia in which the HPV E7 protein reactivates the host DNA replication machinery to support viral DNA replication. In a fraction of the differentiated keratinocytes, E7 also posttranscriptionally induces p21 cip1 , which is distributed in a mutually exclusive manner with unscheduled cellular DNA synthesis. In this study, double immunofluorescence labeling unexpectedly revealed that E7 caused a concordant accumulation of both cyclin E and p21 cip1 to high levels in patient papillomas and in organotypic cultures of primary human keratinocytes. The induction of cyclin E is mutually exclusive with unscheduled cellular DNA synthesis or abundant viral DNA. These novel virus- host interactions in differentiated keratinocytes are in contrast to previous observations made in submerged proliferating cultures, in which HPV E7 induces cyclin E and overcomes p21 cip1 inhibition of S-phase entry. We propose that an appropriately timed induction of cyclin E/cyclin-dependent kinase 2 by HPV E7 in postmitotic cells enables S-phase reentry and HPV DNA amplification, whereas prematurely induced cyclin E stabilizes p21 cip1 protein, which then inhibits cyclin E/ cyclin-dependent kinase 2. Consequently, cyclin E and p21 cip1 both fail to turn over, and DNA synthesis does not occur. Introduction The large family of HPVs 3 is tropic for cutaneous and muco- sal squamous epithelial cells. HPVs induce warty lesions in which viral mRNA transcription, DNA amplification, and progeny virion production are dependent on squamous dif- ferentiation (reviewed in Ref. 1). Because HPV replication requires host DNA replication machinery (2), which is no longer present in postmitotic, differentiated cells (3, 4), the virus reactivates all of the cellular genes necessary to sup- port DNA synthesis. Using organotypic (raft) cultures of PHKs grown at the media-air interface on a dermal equiva- lent raft, we have shown previously that HPV-18 E7 driven by the native differentiation-dependent viral enhancer-promoter accomplishes this task. We demonstrated an induction of unscheduled host DNA synthesis in postmitotic, differenti- ated keratinocytes in raft cultures (5, 6). HPV-18 and HPV-16 are considered to be high-risk viral types because infections can occasionally progress to cancers. Infections by low-risk types such as HPV-6 and HPV-11 virtually never progress to higher-grade lesions, yet unscheduled host DNA synthesis does take place in a fraction of the postmitotic, differentiated cells (5, 6). The distinct pathological properties of these HPV types are attributed to differences in the interactions be- tween the viral E6 and E7 proteins and the tumor suppressor proteins, p53 and the retinoblastoma susceptibility protein (pRB) family of proteins, respectively. In vitro, viral oncopro- teins E7 and E6 from high-risk HPVs can immortalize PHKs (see reviews in Refs. 1 and 7). Cell cycle progression is controlled by cyclins and Cdks (8). The unphosphorylated form of pRB binds to a family of E2F/DP transcription factor complexes and represses the E2F-responsive genes necessary for S-phase entry and pro- gression (for reviews, see Refs. 9, 10; Fig. 7). The D-type cyclins and Cdk4 and Cdk6 phosphorylate and inactivate pRB in response to mitogenic signals. Cyclin E/Cdk2 activity is present briefly in the late G 1 and early S phases and is critical for S-phase entry. Transfection of a cyclin E expres- sion vector is able to speed up entry into the S phase (11–16). In tumor cells in which cyclin D/Cdk4 or Cdk6 is functionally inactivated by the over-expression of inhibitor p16 INK4a , over-expression of cyclin E/Cdk2 results in the phosphoryl- ation of pRB (17). Furthermore, ectopic expression of cyclin E/Cdk2 can drive cells into S phase, despite the presence of a mutated pRB that can no longer be phosphorylated (Refs. 16, 18, and 19; reviewed in Ref. 20). Conversely, depletion of Cdk2 or addition of the p21 cip1 protein inhibits chromosomal Received 11/20/98; revised 1/6/99; accepted 1/6/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. 1 Supported by USPHS Grants CA36200 and DE/CA11910. Y. J. is a recipient of USPHS Training Grant T32 CA09467 in Molecular and Viral Oncology. Partial support for the Digital Imaging Microscopy Facility was provided by a grant from the University of Alabama at Birmingham Health Services Foundation (to T. R. B.) and USPHS P30 AI27767 (to Eric Hunter). 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Genetics, 1918 University Boulevard, McCallum Basic Science Research Building, Room 510, University of Alabama at Birmingham, Birmingham, AL 35294-0005. Phone: (205) 975- 8300; Fax: (205) 975-6075; E-mail: ltchow@uab.edu. 3 The abbreviations used are: HPV, human papillomavirus; Cdk, cyclin- dependent kinase; PHK, primary human keratinocyte; PCNA, proliferating cell nuclear antigen; URR, upstream regulatory region; IF, immunofluo- rescence; BrdUrd, bromodeoxyuridine; CR, conserved region. 101 Vol. 10, 101–111, February 1999 Cell Growth & Differentiation