Human papillomavirus type 16 E6 and HPV‐16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: Roles of p53 and caspase activation

Abstract

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.

Sodium butyrate induced keratinocyte apoptosis

“…Although we were unable to detect an increase in the active caspase 3 fragment by western blot, activation of caspase 3 was confirmed by a reduction in the levels of pro-caspase 3 and cleavage of PARP. Proteolysis of pro-caspase 3 has been directly associated with increased caspase activity in keratinocytes induced to undergo apoptosis by chemotherapeutic agents and UV radiation [46,47]. Moreover, the reduced levels of pro-caspase 3 following UV exposure corresponded to increased PARP cleavage which was reduced by a specific caspase 3 inhibitor [47], consistent with the results shown here (Fig.…”

The HPV-16 E7 Oncogene Sensitizes Malignant Cells to IFN-α-Induced Apoptosis

“…Oncogenic sensitization to apoptosis induced by several signals has been reported previously, (24)(25)(26) as has the ability of some oncogenes to induce apoptosis-insensitive phenotypes. (27,28) One of the first described examples of oncogenic sensitization to apoptosis was the effect of c-Myc overexpression in fibroblasts.…”

Human papillomavirus positive squamous cell carcinoma of the oropharynx

“…30,31 Whether the viral oncoproteins E6 and E7 themselves may render cells more susceptible for DNA damage-induced apoptosis remains controversial. 32,33 Besides radiosensitization by viral proteins, the higher radiocurability of HPV-infected tumors also may be attributed to yet undefined differences in their specific oncogenesis. Finally, parameters related to the specific risk profile of patients with HPV positive oropharyngeal carcinoma, such as the absence of exposure to tobacco and alcohol during radiotherapy as well as patients compliance with treatment, also may contribute to the higher tumor control rates in these individuals.…”