Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation.
OBJECTIVE
Low-dose-rate radiation therapy has been widely used in the treatment of urogenital malignancies. When continuously exposed to low-dose-rate ionizing radiation, target cancer cells typically exhibit abnormalities in replicative cell-cycle progression. Cancer cells that arrest in the G2 phase of the cell cycle when irradiated may become exquisitely sensitive to killing by further low-dose-rate radiation treatment. Oncogenic human papillomaviruses (HPVs), which play a major role in the pathogenesis of uterine cervix cancers and other urogenital cancers, encode E6 and E7 transforming proteins known to abrogate a p53-dependent G1 cell-cycle checkpoint activated by conventional acute-dose radiation exposure. This study examined whether expression of HPV E6 and E7 oncoproteins by cancer cells alters the cell-cycle redistribution patterns accompanying low-dose-rate radiation treatment, and whether such alterations in cell-cycle redistribution affect cancer cell killing.


METHODS
RKO carcinoma cells, which contain wild-type P53 alleles, and RKO cell sublines genetically engineered to express HPV E6 and E7 oncoproteins, were treated with low-dose-rate (0.25-Gy/h) radiation and then assessed for p53 and p21WAF1/CIP1 polypeptide induction by immunoblot analysis, for cell-cycle redistribution by flow cytometry, and for cytotoxicity by clonogenic survival assay.


RESULTS
Low-dose-rate radiation of RKO carcinoma cells triggered p53 polypeptide elevations, p21WAF1/CIP1 induction, and arrest in the G1 and G2 phases of the cell cycle. In contrast, RKO cells expressing E6 and E7 transforming proteins from high-risk oncogenic HPVs (HPV 16) arrested in G2, but failed to arrest in G1, when treated with low-dose-rate ionizing radiation. Abrogation of the G1 cell-cycle checkpoint activated by low-dose-rate radiation exposure appeared to be a characteristic feature of transforming proteins from high-risk oncogenic HPVs: RKO cells expressing E6 from a low-risk nononcogenic HPV (HPV 11) exposed to low-dose-rate radiation arrested in both G1 and G2. Surprisingly, despite differences in cell-cycle redistribution accompanying low-dose-rate radiation treatment associated with high-risk HPV transforming protein expression, no consistent differences in clonogenic survival following low-dose-rate radiation treatment were found for RKO cell sublines expressing high-risk HPV oncoproteins and arresting only in G2 during low-dose-rate radiation exposure vs. RKO cell sublines exhibiting both G1 and G2 cell-cycle arrest when irradiated.


CONCLUSIONS
The results of this study demonstrate that neither HPV oncoprotein expression nor loss of the radiation-activated G1 cell-cycle checkpoint alter the sensitivity of RKO carcinoma cell lines to low-dose-rate radiation exposure in vitro. Perhaps for urogenital malignancies associated with oncogenic HPVs in vivo, HPV oncoprotein-mediated abrogation of the G1 cell-cycle checkpoint may not limit the potential efficacy of low-dose-rate radiation therapy.
DeWeese
T L
TL
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Walsh
J C
JC
Dillehay
L E
LE
Kessis
T D
TD
Hedrick
L
L
Cho
K R
KR
Nelson
W G
WG
D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D016193:G1 Phase / Q000528:radiation effects*; D016195:G2 Phase / Q000528:radiation effects*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D011829:Radiation Dosage; D011836:Radiation Tolerance; D012097:Repressor Proteins; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism* / Q000528:radiation effects
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16