Induction of virus-producing tumours in athymic nude mice by bovine papillomavirus type 4.

Bovine papillomavirus type 4 (BPV-4), the causative agent of alimentary papillomatosis, has been used to infect, in vitro, fragments of palatine mucosa from late term bovine fetuses. These small explants were placed beneath the renal capsule of athymic nude mice where they grew to produce, at first, squamous epithelial cysts containing BPV-4 genomic DNA and, later, papillomas which were morphologically identical to those of cattle and which contained large amounts of replicating virus. The possible utility of this technique in assessing neutralising antibodies in vaccine development is discussed.

Studies on vaccination against papillomaviruses: the immunity after infection and vaccination with bovine papillomaviruses of different types.

Calves, free of antibodies to bovine papillomaviruses (BPV), were reared in isolation. One was infected with BPV-2, developed tumours and was resistant to homologous reinfection. Groups of calves were infected with BPV-2, BPV-5 or BPV-6; they all developed and subsequently rejected type-specific tumours. They were then infected with BPV-4; they were not immune and oral papillomas were induced. Groups of animals were vaccinated by intramuscular preparations of purified BPV-4 and BPV-6 and were challenged with homologous virus; all were immune to reinfection. An earlier experiment had shown this to be true for BPV-2. Two calves, immune to BPV-6, were not immune to BPV-1. These experiments, although they do not cover all the possibilities of reciprocal immunisation and challenge, indicate that prophylactic immunity to a range of papillomaviruses is type-specific. This is the first clear demonstration of this phenomenon in the papillomavirus group.

Studies on vaccination against papillomaviruses: a comparison of purified virus, tumour extract and transformed cells in prophylactic vaccination.

Calves were vaccinated with two preparations made from one cutaneous fibropapilloma induced by bovine papillomavirus type 2 (BPV-2). One vaccine consisted of homogenised tumour; the other contained purified virus only. Both produced resistance to a heavy challenge infection of BPV-2. One calf in the vaccinated group developed a small tumour and rejected it earlier than the control calves. It would appear likely that the prophylactic immune response was induced by viral structural proteins only and that tumour-specific antigens are unnecessary. Bovine fibroblasts were transformed in vitro by BPV-2 and administered as a vaccine; immunity was not induced.

Protection of cells from methotrexate toxicity by 7-hydroxymethotrexate.

Cell growth survival studies have revealed that 7-OH methotrexate is two orders of magnitude less cytotoxic to human melanoma and human acute lymphoblastic leukaemia (ALL) cells in vitro than methotrexate. The influence of 7-OH methotrexate on methotrexate toxicity was investigated by studying cell growth in the presence of methotrexate and its 7-OH metabolite and by studying [3H]-methotrexate movement across the plasma membrane of isolated human cells. Transport was followed for net entry of the drug into drug-free cells, net exit of drug into drug-free medium and for unidirectional exit fluxes with drug and/or metabolite in the extracellular medium (exchange exit). Results indicate that 7-OH methotrexate (10(-6) M) interacts with melanoma cells to reduce the initial cellular uptake rate of [3H]-methotrexate but that no such interaction occurs with ALL cells. Efflux measurements revealed that a stimulatory effect of extracellular methotrexate on [3H]-methotrexate exit was apparent and that extracellular 7-OH methotrexate had a less stimulatory effect. Overall, loss of intracellular drug was greater from melanoma cells than from ALL cells. The results suggest that the drug resistance encountered following high dose therapy may be due to reduced cellular uptake and/or increased efflux of methotrexate from cells, both events being enhanced by 7-OH methotrexate. In addition, there is an apparently endogenous resistance of the melanomas to methotrexate as regards time of exposure to this agent which could also contribute to the lack of clinical response when compared to ALL.

Cooperation between papillomavirus and chemical cofactors in oncogenesis.

Papillomaviruses have been identified as causative agents of squamous cell carcinoma in humans and other mammals, and several papillomavirus-encoded proteins have been found to possess transforming activity. However, papillomavirus infection per se appears insufficient to elicit carcinomas in the majority of cases, and other agents have been implicated as cofactors. This review presents epidemiological studies linking environmental carcinogens to papillomavirus-associated carcinoma, and evidence from experimental systems that attempt to identify the targets of carcinogens and viral oncoproteins, and the interactions of both in progression to carcinoma.

Cytotoxicity of etretinate and vindesine.

The effects of an aromatic retinoid, etretinate and a vinca alkaloid, vindesine were investigated by culture of malignant melanoma cells in vitro with these two agents; either separately or in combination. Etretinate inhibited growth of a murine melanoma but only minimal effects were recorded with two human melanomas. Vindesine however, was inhibitory for all of the cell lines and this effect was enhanced in the presence of the retinoid. Entry of 3H labelled vindesine or etretinate into drug free cells was followed in the absence or presence of unlabelled drug. It was found that etretinate enhanced cellular uptake of vindesine in two of the cell lines and this may be responsible for the enhanced toxicity of vindesine in the presence of etretinate. The human melanoma which did not exhibit retinoid stimulated vindesine uptake, appeared to be intrinsically sensitive to the vinca alkaloid. No effect on cellular retinoid uptake by vindesine was recorded in any of the melanomas. The results indicate that the intracellular concentrations combined with the intrinsic sensitivities of each cell line to etretinate and vindesine determines the toxic response.

Interaction between bovine papillomavirus type 4 and cocarcinogens in the production of malignant tumours.

Bovine fetal palatine tissue infected with bovine papillomavirus type 4 (BPV-4) was implanted subcutaneously in athymic nude mice. The implants developed into cysts containing papillomas essentially the same as those in the natural host. In order to investigate the interaction of cocarcinogens with BPV-4 in cell transformation, the virus-infected implants were exposed in vivo to either the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the tumour initiator 7,12-dimethylbenz[a]-anthracene (DMBA). Papillomas were detected in a greater number of infected implants in the presence of either TPA or DMBA than in the absence of either of these chemicals indicating interaction between the virus and these two agents. Moreover, malignantly transformed cells arose with high frequency from infected implants that had been exposed to either chemical. In the presence of chemical and absence of virus or vice versa no neoplastic changes were seen histologically, indicating that cooperation between virus and cocarcinogen is required for transformation.

Paradoxical response of malignant melanoma to methotrexate in vivo and in vitro.

Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.

Experience with the colony-forming (stem cell) assay of in vitro chemosensitivity in the management of patients with advanced malignant melanoma.

Twenty-two patients with advanced malignant melanoma had tumor nodules biopsied and tumor cell colonies established in vitro in the colony-forming assay in the presence of vindesine. The patients then received iv vindesine at a dose of 3 mg/m2/week for 6 weeks. In four patients, in vitro death of cells exposed to vindesine in vitro correlated with clinical response to therapy. In the remaining 18 patients, lack of both in vitro and clinical response was noted.

Vaccination of cattle with bovine papillomavirus type 4 L2 elicits the production of virus-neutralizing antibodies.

Prophylactic vaccination of cattle with the N terminus (L2a, aa 11-200) of the minor capsid protein L2 completely prevented bovine papillomavirus type 4 (BPV-4) infection of the alimentary canal. To investigate the mechanisms underlying protection from viral infection, sera from vaccinated animals were analysed in neutralization assays both in the nude mouse xenograft system and in cattle. BPV-4 retained its infectivity when incubated with preimmune cattle sera, whereas, when incubated with immune sera from animals vaccinated with either whole L2 or its N terminus L2a, its infectivity was greatly reduced, indicating that the immune sera had neutralizing activity against the virus. This activity could be abrogated by absorbing the immune sera with L2 or L2a, thus indicating that virus neutralization was due to the presence in the immune sera of anti-L2 antibodies.

Malignant transformation of a papilloma induced by bovine papillomavirus type 4 in the nude mouse renal capsule.

A papillomatous cyst was induced by implanting bovine foetal palate epithelium, infected in vitro with bovine papillomavirus type 4 (BPV-4), beneath the renal capsule of a nude mouse. The benign tumour underwent malignant progression, developing into a squamous cell carcinoma with metastatic deposits in the spleen. The bovine origin of both the renal and splenic cancers was confirmed by the presence of bovine major histocompatibility complex class I antigens in the cancer cells and by sequencing the Harvey-ras 1 gene, which was shown to be of bovine origin. BPV-4 DNA was present in the residual papillomatous fronds of the renal cancer, but was absent from the carcinoma proper and for the splenic metastasis. These results confirm that BPV-4 is a carcinogenic agent and that its genetic information is not necessary for the maintenance of the malignant phenotype. Moreover the system provides the opportunity to investigate the role of viral and chemical carcinogens in an experimental system.

Studies on vaccination against papillomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins.

The L1 and L2 proteins of BPV-2 have been produced in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins have been used to vaccinate calves both prophylactically and therapeutically. The L1 fusion protein prevented tumor formation when administered before challenge with BPV-2, while the L2 fusion protein was very effective in promoting tumor rejection, independently from whether it was administered before or after challenge. Animals vaccinated with L1, but not with L2, responded rapidly with production of serum neutralizing antibodies, showing that this peptide contains B-cell-specific epitopes. The massive infiltration of lymphocytes in the tumors of L2-vaccinated animals suggests that the peptide contains epitopes specific for T-cells. The two structural proteins of BPV-2 therefore interact with both efferent arms of the immune system, and this observation allows the choice between two different types of antiviral vaccination.