Hang Yuan

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter

The papillomavirus E6 protein binds and directs the ubiquitin-dependent degradation of the p53 tumor suppressor protein. Independent of this p53-degradative function, however, E6 induces cellular telomerase activity. This increase in enzyme activity reflects E6-enhanced transcription of the human telomerase reverse transcriptase (hTERT) catalytic subunit, but the molecular basis for this transactivation is unknown. In the present study, we demonstrate that E6/Myc interactions regulate hTERT gene expression. Mad protein, a specific antagonist of Myc, repressed E6-mediated transactivation of the hTERT promoter and this repression was relieved by Myc overexpression. The proximal Myc/ Max-binding element (E-box) in the hTERT promoter was the major determinant of both E6 and Myc responsiveness in keratinocytes. E6 did not alter Myc protein expression or Myc/Max association, and the induction of hTERT by Myc/E6 was independent of Myc phosphorylation at Thr-58/Ser-62 within the transactivation domain. However, immunoprecipitation studies demonstrated that endogenous Myc protein coprecipitated with E6 protein and chromatin immunoprecipitation analyses demonstrated that both E6 and Myc proteins bound to a minimal 295-bp hTERT promoter. Only the “high-risk” E6 proteins bound to the hTERT promoter, consistent with their preferential ability to induce telomerase. The observation that E6 associates with Myc complexes and activates a Myc-responsive gene identifies a mechanism by which this oncogene can modulate cell proliferation and differentiation.

Immunization with a Pentameric L1 Fusion Protein Protects against Papillomavirus Infection

ABSTRACT The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathioneS-transferase (GST) fusion protein in Escherichia coli and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.

Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air–liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Activation of the Canonical Wnt Pathway during Genital Keratinocyte Transformation: A Model for Cervical Cancer Progression

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic beta-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from beta-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of beta-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of beta-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.

Dihydroartemisinin Is Cytotoxic to Papillomavirus-Expressing Epithelial Cells In vitro and In vivo

Nearly all cervical cancers are etiologically attributable to human papillomavirus (HPV) infection and pharmaceutical treatments targeting HPV-infected cells would be of great medical benefit. Because many neoplastic cells (including cervical cancer cells) overexpress the transferrin receptor to increase their iron uptake, we hypothesized that iron-dependent, antimalarial drugs such as artemisinin might prove useful in treating HPV-infected or transformed cells. We tested three different artemisinin compounds and found that dihydroartemisinin (DHA) and artesunate displayed strong cytotoxic effects on HPV-immortalized and transformed cervical cells in vitro with little effect on normal cervical epithelial cells. DHA-induced cell death involved activation of the mitochondrial caspase pathway with resultant apoptosis. Apoptosis was p53 independent and was not the consequence of drug-induced reductions in viral oncogene expression. Due to its selective cytotoxicity, hydrophobicity, and known ability to penetrate epithelial surfaces, we postulated that DHA might be useful for the topical treatment of mucosal papillomavirus lesions. To test this hypothesis, we applied DHA to the oral mucosa of dogs that had been challenged with the canine oral papillomavirus. Although applied only intermittently, DHA strongly inhibited viral-induced tumor formation. Interestingly, the DHA-treated, tumor-negative dogs developed antibodies against the viral L1 capsid protein, suggesting that DHA had inhibited tumor growth but not early rounds of papillomavirus replication. These findings indicate that DHA and other artemisinin derivatives may be useful for the topical treatment of epithelial papillomavirus lesions, including those that have progressed to the neoplastic state.

The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein.

Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.

Simian Virus 40 Small Tumor Antigen Activates AKT and Telomerase and Induces Anchorage-Independent Growth of Human Epithelial Cells

ABSTRACT Human keratinocytes immortalized by full-length or early-region simian virus 40 (SV40) DNA grow in agarose and form tumors in nude mice, in contrast to keratinocytes immortalized by the E6/E7 genes of human papillomaviruses. To determine the molecular basis for this biological difference in growth, we have used the individual SV40 oncogenes (large T antigen [LT] and small t antigen [st]) and human papillomavirus oncogenes (E6/E7) to study the progression of human epithelial cells from the nonimmortal to the immortal state as well as from the immortal to the anchorage-independent state. Transfection of primary human foreskin keratinocytes with LT did not immortalize cells but did extend the in vitro life span and produced cells that were resistant to calcium- and serum-induced terminal differentiation. Cells transfected with st alone did not passage beyond vector-transfected keratinocytes. The simultaneous expression of LT- and st-immortalized keratinocytes occurred without evidence of crisis and, as anticipated, these immortal cells were anchorage- independent for growth. Interestingly, we found that keratinocytes expressing both LT and st, but not keratinocytes with LT alone, exhibited increased phosphorylation of the protein kinase AKT. In addition, AKT activation was paralleled by an increase in telomerase activity. Addition of st to anchorage-dependent keratinocytes, expressing either LT (nonimmortal) or E6/E7 (immortal), converted the cells to anchorage independence, with similar accompanying increases in AKT phosphorylation and telomerase activity. However, it was not possible to induce keratinocyte growth in agarose with activated AKT and/or overexpressed hTERT, indicating that these newly defined st-induced activities are not sufficient for progression to the anchorage-independent state.

Severe Papillomavirus Infection Progressing to Metastatic Squamous Cell Carcinoma in Bone Marrow-Transplanted X-Linked SCID Dogs

ABSTRACT Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain (γc) gene and is identical clinically and immunologically to human XSCID, making it a true homologue of the human disease. Bone marrow-transplanted (BMT) XSCID dogs not only engraft donor T cells and reconstitute normal T-cell function but, in contrast to the majority of transplanted human XSCID patients, also engraft donor B cells and reconstitute normal humoral immune function. Shortly after our initial report of successful BMT of XSCID dogs, it soon became evident that transplanted XSCID dogs developed late-onset severe chronic cutaneous infections containing a newly described canine papillomavirus. This is analogous to the late-onset cutaneous papillomavirus infection recently described for human XSCID patients following BMT. Of 24 transplanted XSCID dogs followed for at least 1 year post-BMT, 71% developed chronic canine papillomavirus infection. Six of the transplanted dogs that developed cutaneous papillomas were maintained for >3 1/2 years post-BMT for use as breeders. Four of these six dogs (67%) developed invasive squamous cell carcinoma (SCC), with three of the dogs (75%) eventually developing metastatic SCC, an extremely rare consequence of SCC in the dog. This finding raises the question of whether SCC will develop in transplanted human XSCID patients later in life. Canine XSCID therefore provides an ideal animal model with which to study the role of the γc-dependent signaling pathway in the response to papillomavirus infections and the progression of these viral infections to metastatic SCC.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.