Evaluation of positron emission tomographic tracers for imaging of papillomavirus-induced tumors in rabbits.

In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2-18F-fluoro-d-glucose (18F-FDG), 11C-choline, and 18F-fluorothymidine (18F-FLT) to detect papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers 11C-choline and 18F-FLT of tumors and reference tissues were examined relative to the clinical standard, 18F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with 18F-FDG and 11C-choline, whereas 18F-FLT failed to detect the tumors at all measured time points. Our data show that the PET tracer 18F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to 11C-choline and 18F-FLT. However, 11C-choline imaging, which has previously been applied to detect various tumor entities in patients, appears to be an alternative to 18F-FDG.

A recombinant cottontail rabbit papillomavirus genome for ectopic expression of genes in cells infected with virus in vivo.

The objective of this study was to construct a cottontail rabbit papillomavirus (CRPV) genome that would co-express a gene of choice and the viral genome simultaneously. Using this construct, the effects of the ectopic expression of diverse viral or cellular genes on PV-infected cells can be examined to elucidate which genes are essential for tumor formation. CRPV-pLAIIdelXba1, which lacks the major portion of L2 (designated the XbaI fragment), has been previously shown to fully retain the ability to induce tumors, and this ability was confirmed in this study. Insertion of the XbaI fragment in an antisense orientation did not change the efficiency of tumor induction. An SV40 overexpression cassette that originated from pSG5 and contains a more diverse multiple cloning site (MCS) was cloned into CRPV-Xba1-mcs, a CRPV genome based on CRPV-pLAIIdelXba1 that contains an additional MCS inserted via XbaI digestion. Additionally, the L1 ATG initiation codon of this construct, designated CRPV-Xba1-oe-WT, was mutated to avoid unnecessary L1 protein expression, which produced the CRPV-Xba1-oe-L1mut construct. Injection of these constructs into two New Zealand White rabbits and monitoring of tumor growth for two to six months showed that CRPV-Xba1-oe-WT induced tumors at 1/10 and 1/10 of the injection sites in two animals, while the control injections in each rabbit induced tumors at 3/10 and 4/10 injection sites, respectively. However, CRPV-Xba1-oe-L1mut induced tumors at 3/10, 6/10, 7/12 and 11/12 sites in four injected animals, and the control injections induced tumor growth in these animals at 6/10, 10/10, 12/12 and 12/12 of the injected sites, respectively. Thus, CRPV-Xba1-oe-L1mut could potentially be used to conduct overexpression experiments in vivo that can be used to measure the negative or positive influences of ectopically expressed foreign or HPV genes on tumor growth.

Cutaneous papillomavirus E6 proteins must interact with p300 and block p53-mediated apoptosis for cellular immortalization and tumorigenesis.

The binding of the papillomavirus E6 protein to E6AP and the induction of p53 degradation are common features of high-risk genital human papillomaviruses (HPV); cutaneous HPVs, on the other hand, lack these capacities. Nevertheless, several cutaneous HPV types of the beta-genus, such as HPV38 are associated with tumor formation when combined with genetic predisposition, immunosuppression, or UV exposure. In an animal model system, the cottontail rabbit papillomavirus (CRPV) rapidly induces skin cancer without additional cofactors, and CRPVE6 and E7 immortalize rabbit keratinocytes in vitro. However, CRPVE6 neither interacts with E6AP and p53 nor does it induce p53 degradation. In this study, we show that the interaction of CRPVE6, or HPV38E6, with the histone acetyltransferase p300 is crucial to inhibit the ability of p53 to induce apoptosis. Strikingly, E6 mutants deficient for p300 binding are incapable of preventing p53 acetylation, p53-dependent transcription, and apoptosis induction. Moreover, E6 mutants deficient for p300 binding cannot contribute to HPV38-induced immortalization of human keratinocytes or CRPV-induced tumor formation. Our findings highlight changes in the p53 acetylation status mediated by the viral E6 protein as a crucial requirement in the ability of high-risk cutaneous papillomaviruses to immortalize primary keratinocytes and induce tumors. Cancer Res; 70(17); 6913-24. (c)2010 AACR.

Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes.

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for approximately 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP.

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.