HPV-16 E6/7 Immortalization Sensitizes Human Keratinocytes to Ultraviolet B by Altering the Pathway from Caspase-8 to Caspase-9-dependent Apoptosis*

UVB from solar radiation is both an initiating and promoting agent for skin cancer. We have found that primary human keratinocytes undergo an apoptotic response to UVB. To determine whether these responses are altered during the course of immortalization, we examined markers of apoptosis in primary human foreskin keratinocytes (HFK) transduced with either a retroviral vector expressing the E6 and E7 genes of HPV-16 or with empty vector alone (LXSN-HFK). Whereas LXSN-HFK as well as early passage keratinocytes expressing HPV-16 E6 and E7 (p7 E6/7-HFK) were both moderately responsive to UVB irradiation, late passage-immortalized keratinocytes (p27 E6/7-HFK) were exquisitely sensitive to UVB-induced apoptosis. After exposure to UVB, enhanced annexin V-positivity and internucleosomal DNA fragmentation were observed in p27 E6/7-HFK compared with either LXSN- or p7 E6/7-HFK. Caspase-3 fluorometric activity assays as well as immunoblot analysis with antibodies to caspase-3 and poly(ADP-ribose) polymerase revealed elevated caspase-3 activity and processing at lower UVB doses in p27 E6/7-HFK compared with LXSN- or p7 E6/7-HFK. In addition, the caspase inhibitor DEVD-CHO reduced the apoptotic response and increased survival of all three HFK types. Immunoblot analysis revealed that caspase-8 was activated in all three cell types, but caspase-9 was only activated in p27 E6/7-HFK. Cell cycle analysis further showed that only p27 E6/7-HFK exhibit G2/M accumulation that is enhanced by UVB treatment. This accumulation was associated with a rapid down-regulation of Bcl-2 in these cells. The immortalization process subsequent to the expression of HPV E6 and E7 may therefore determine UVB sensitivity by switching the mode of apoptosis from a caspase-8 to a Bcl-2-caspase-9-mediated pathway of apoptosis.

[1]  T. Jacks,et al.  Sunburn and p53 in the onset of skin cancer , 1994, Nature.

[2]  T. Veldman,et al.  Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein , 2001, Journal of Virology.

[3]  R. Tarone,et al.  Frequent clones of p53-mutated keratinocytes in normal human skin. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Korsmeyer,et al.  Involvement of Microtubules in the Regulation of Bcl2 Phosphorylation and Apoptosis through Cyclic AMP-Dependent Protein Kinase , 1998, Molecular and Cellular Biology.

[5]  D. Brash,et al.  Ultraviolet radiation induced signature mutations in photocarcinogenesis. , 1999, The journal of investigative dermatology. Symposium proceedings.

[6]  J. McDougall,et al.  Telomerase activation by the E6 gene product of human papillomavirus type 16 , 1996, Nature.

[7]  S. Stratton.  Prevention of non-melanoma skin cancer , 2001, Current oncology reports.

[8]  V. Fadok,et al.  Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. , 1992, Journal of immunology.

[9]  F. Pontén,et al.  Persistent p53 mutations in single cells from normal human skin. , 2001, The American journal of pathology.

[10]  Miller Ad,et al.  Improved Retroviral Vectors for Gene Transfer and Expression , 1989 .

[11]  B. Monia,et al.  Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli , 1999, Oncogene.

[12]  C. Meijer,et al.  Non‐random allelic losses at 3p, 11p and 13q during HPV‐mediated immortalization and concomitant loss of terminal differentiation of human keratinocytes , 1998, International journal of cancer.

[13]  Shai Shaham,et al.  The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme , 1993, Cell.

[14]  W. Hahn,et al.  Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics , 2000, Molecular and Cellular Biology.

[15]  E. de Villiers,et al.  Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients. , 1996, Journal of the National Cancer Institute.

[16]  C. Simbulan-Rosenthal,et al.  Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway. , 1998, The Journal of investigative dermatology.

[17]  C. Suschek,et al.  Nitric Oxide Fully Protects against UVA-induced Apoptosis in Tight Correlation with Bcl-2 Up-regulation* , 1999, The Journal of Biological Chemistry.

[18]  I J Christensen,et al.  Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample-storage, staining and internal standardization. , 1983, Cytometry.

[19]  P. Fuchs,et al.  The presence of antibodies against virus‐like particles of epidermodysplasia verruciformis‐associated humanpapillomavirus type 8 in patients with actinic keratoses , 2000, The British journal of dermatology.

[20]  Thomas A. Luger,et al.  Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L , 1998, The Journal of cell biology.

[21]  E. Fuchs,et al.  Bcl-x expression influences keratinocyte cell survival but not terminal differentiation. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[22]  T. Kiyono,et al.  Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells , 1998, Nature.

[23]  C. Meijer,et al.  Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line. , 2001, Journal of the National Cancer Institute.

[24]  Y. Aragane,et al.  Ultraviolet-B-induced apoptosis of keratinocytes: evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells. , 1995, The Journal of investigative dermatology.

[25]  A. Chinnaiyan,et al.  Signal Transduction by DR3, a Death Domain-Containing Receptor Related to TNFR-1 and CD95 , 1996, Science.

[26]  T. Mak,et al.  TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation. , 1999, Journal of immunology.

[27]  E. Stanbridge,et al.  Evidence for a putative senescence gene locus within the chromosomal region 10p14-p15. , 2001, Cancer research.

[28]  F. Zunino,et al.  Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells , 2001, Oncogene.

[29]  D. Zelterman,et al.  Escaping the stem cell compartment: Sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Reutelingsperger,et al.  Expression on B Cells Undergoing Apoptosis Annexin V for Flow Cytometric Detection of Phosphatidylserine , 2022 .

[31]  R. Schlegel,et al.  Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16 , 1996, Journal of virology.

[32]  Miranda Thomas,et al.  The role of the E6-p53 interaction in the molecular pathogenesis of HPV , 1999, Oncogene.

[33]  D. Lowy,et al.  Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity , 1992, Journal of virology.

[34]  B. Nickoloff,et al.  Sunlight-induced basal cell carcinoma tumor cells and ultraviolet-B-irradiated psoriatic plaques express Fas ligand (CD95L). , 1998, The Journal of clinical investigation.