Characterization of DoTc2 4510—Identifying HPV16 Presence in a Cervical Carcinoma Cell Line Previously Considered to Be HPV-Negative

SIMPLE SUMMARY: Cervical cancer is one of the most deadly cancers for women worldwide. Its incidence is, in virtually all cases, causally tied to a human papillomavirus (HPV) infection that had not been cleared by the immune response of the host. There is a small subset of cervical cancers, however, that do not show evidence of HPV presence, thus representing a rare gynecological cancer entity. While HPV-related cervical cancer is studied extensively, HPV-independent cervical cancer studies are lacking, together with appropriate models. We obtained a cervical cancer cell line, DoTc2 4510, described in the literature as HPV-negative. After suspicous preliminary results showing HPV presence, we proceeded to characterize the cell line in detail, proving it is truly HPV-transformed. With this publication, we aim to inform the community about the HPV status of the studied cell line, as well as emphasize the importance of stringent controls in cancer model establishment, especially for rare cancers. ABSTRACT: Cervical cancer is the fourth leading cause of cancer deaths in women, with over 340,000 women dying from this disease in 2020. Almost all cases have an underlying persistent infection with an oncogenic high-risk type of human papillomavirus (HPV), mainly HPV16. While cervical squamous cell carcinoma is hardly ever HPV-negative, a small subset of adenocarcinoma exhibits absence of HPV, even after disproval of false-negative testing results due to low viral load. This proportion is evident in many cervical cancer studies and is reflected in the repertoire of model cell lines commonly used in research. As the viral origin of cervical cancer makes it a disease preventable and potentially treatable by immunotherapeutic approaches, it is the focus of many studies. For pertinent research, both a broad set of HPV-infected cervical carcinoma models are required, as well as stringent negative controls. A ubiquitously used HPV-negative cervical adenocarcinoma cell line is C-33A. Another cervical cancer cell line is available for purchase from the American Type Culture Collection (ATCC), namely DoTc2 4510, described to be HPV-negative and thus as a model for a rare gynecological malignancy. Here, we present findings proving that DoTc2 4510 is, in fact, an HPV16-positive cell line. This we assessed using a highly sensitive nested multiplex PCR protocol adapted for the identification of 12 carcinogenic HPV types and a second PCR targeting the HPV16 oncogenes E6 and E7. Subsequently, the protein expression of E6 and E7 was examined, as well as the expression of their target proteins p53, p21, and p16(INK4a), to assess E6/E7 functionality. Finally, to attest to the survival dependence of DoTc2 4510 cells on HPV16, we performed an HPV16 E6/E7-targeted siRNA knock-down, which indeed led to senescence induction. Together, these findings demonstrate that DoTc2 4510 is an HPV16-transformed cell line.