Extracts Of Papillomas Research Articles

PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells

Laryngeal papillomas are caused by infection of the laryngeal epithelium by human papillomavirus type 6 or type 11 (HPV-6/-11). Previous studies in our laboratory have demonstrated an increase in PI3 kinase levels in papilloma tissue. However, activation of the downstream effector of PI3 kinase, protein kinase B (PKB/Akt), was reduced. This observation was explained by the elevated expression of the phosphatase and tensin homologue (PTEN), a recently characterized tumour suppressor, in papilloma tissue. Recent investigation of the possible functional roles of PTEN during papilloma development has now indicated that the level of tyrosine(705)-phosphorylated signal transducer and activator of transcription 3 [PTyr(705)STAT3] could be inversely correlated to that of PTEN as well. In vitro phosphatase assays suggested the presence of an increased level of a PTyr(705)STAT3 phosphatase in papilloma extract. Immunodepletion of PTEN from papilloma extracts resulted in a reduction of the PTyr(705)STAT3 phosphatase activity. Transfection of PTEN cDNA into HeLa cells attenuated STAT3 phosphorylation at Tyr(705) in a dose-dependent manner. This attenuation of STAT3 phosphorylation was independent of the STAT3 kinase. Interestingly, introduction of a lipid phosphatase mutant of PTEN (G129E) resulted in heightened PTyr(705)STAT3 phosphatase activity, relative to that obtained from wild-type PTEN transfection. These data indicate that PTEN negatively regulates STAT3 activation in HPV-infected papilloma cells. Induction of PTEN and reduction of activated STAT3 might be a result of a host defence mechanism or a virus-directed strategy to alter normal epithelial differentiation programming.

Activation of mouse epidermal tumor ornithine decarboxylase by GTP: evidence for different catalytic forms of the enzyme.

In crude extracts of epidermal papillomas induced by an initiation-promotion protocol, ornithine decarboxylase (OrnDCase) activity was increased by the addition of GTP to the enzyme assay. No effect of GTP on the phorbol ester-induced enzyme isolated from normal epidermis was observed. Kinetic analyses indicated that the major effect of the nucleotide on the tumor-derived enzyme was to lower the apparent Km for L-ornithine. When papilloma OrnDCase was partially purified by gel-filtration chromatography, two forms of the enzyme were resolved, only one of which was found in an epidermal extract from phorbol 12-myristate 13-acetate-treated mice. The enzymatic properties of the two forms of papilloma enzyme were compared. The higher molecular weight form (peak I) was activated by GTP, while the lower molecular weight form (peak II) was not. As expected from the kinetic analyses of the crude papilloma extracts, the apparent Km of peak I enzyme for L-ornithine was very high (1.25 mM) but was much lower in the presence of GTP (0.02 mM). The two forms of papilloma OrnDCase differed in their sensitivities to heat inactivation and the ability of GTP to protect against heat inactivation. The K1/2 for activation of peak I OrnDCase by GTP was 0.1 microM. The activation process was irreversible and did not require Mg2+. When several nucleotides were tested for their ability to activate peak I OrnDCase, only GTP, dGTP, and the nonhydrolyzable derivative GTP[gamma-S] were effective, while GDP, GMP, ATP, and CTP were relatively ineffective. Our results demonstrated the existence of two forms of OrnDCase in epidermal tumor extracts, of which one can be activated by GTP and one cannot. The significance of these findings for the regulation of this enzyme in normal and tumor cells is discussed.

Ornithine decarboxylase from mouse epidermis and epidermal papillomas: differences in enzymatic properties and structure.

The properties of ornithine decarboxylase (OrnDCase) from mouse epidermis and benign epidermal tumors (papillomas) induced by the initiation-promotion protocol were compared. When crude extracts from each tissue were incubated at 55 degrees C, epidermal OrnDCase was rapidly inactivated, but the papilloma OrnDCase was more heat stable. Each of five individual papilloma extracts contained OrnDCase activity that was considerably more resistant to heat inactivation than was epidermal OrnDCase. Mixing of a papilloma and epidermal extract produced an intermediate heat-inactivation profile, suggesting that the differences in OrnDCase heat stability are not due to non-OrnDCase components of the extracts. Kinetic analyses indicated that the papilloma OrnDCase has an altered affinity for its substrate, L-ornithine, compared to epidermal OrnDCase. The apparent Km for L-ornithine for the epidermal enzyme was 0.07 mM while the Km values for the individual papilloma OrnDCases clustered around two higher values, 0.3 mM and 1.0 mM. The papilloma OrnDCases, but not epidermal OrnDCase, were activated by GTP and to a lesser extent by CTP. Immunoblot analysis showed the existence of multiple forms of OrnDCase in both epidermis and papilloma that differed in isoelectric point but not subunit molecular weight. None of the species of OrnDCase present in the epidermal extract coincided with the species present in papilloma. These results suggest that one consequence of neoplastic transformation in this in vivo system is the presence of an OrnDCase protein in benign tumors that differs structurally and functionally from the OrnDCase present in normal epidermis. The possible mechanisms responsible for these results and their significance for neoplastic development in this tissue are discussed.

New antigens in hamster embryo cells transformed in vitro by bovine papilloma extracts.

BOVINE papilloma virus is known1–3 to induce the transformation of cell cultures of various origins.

Urea-extractable antigens in normal, benign, and neoplastic mouse epidermis.

Antisera were prepared in rabbits against urea-extractable proteins of normal mouse epidermis, papillomas, and squamous-cell carcinomas. The relationship of these antisera and the antigens of the three tissues was studied by immunodiffusion in agar before and after inhibition treatment of these antisera with the three antigens. These procedures revealed that (1) the epidermis has urea-extractable antigens which are present in much smaller amounts in the urea extracts of papillomas and squamous-cell carcinomas, and (2) papillomas and carcinomas have several urea-extractable antigens in common, and both the benign and malignant growths each have at least one antigen present in greater amount in one tissue than in the other. The papilloma and carcinoma antigens are present, but in small amounts in normal epidermis.

INFLUENCE OF EXTRANEOUS PROTEIN AND VIRUS CONCENTRATION ON THE INACTIVATION OF THE RABBIT PAPILLOMA VIRUS BY X-RAYS

The pronounced resistance to the x-rays manifested by the papilloma virus in ordinary suspensions is due to the protecting influence of extraneous matter and also in considerable degree to the amount of virus present in the preparation. Two to 4 million r were required to inactivate the virus contained in the crude papilloma extracts prepared for the present work, whereas 100,000 r or less was enough to inactivate comparable concentrations of virus after extraneous matter had been excluded by repeated differential centrifugation. The addition of normal rabbit serum or crystalline egg albumin to purified suspensions of virus was found to increase greatly the amount of irradiation required to inactivate the virus. Furthermore the percentage destruction of virus by a given amount of irradiation increases as the concentration is decreased by dilution with saline or buffer solutions. As little as 3,000 r will inactivate much of the virus in very dilute suspensions. The complement-binding antigen of papilloma virus suspensions is also inactivated by x-rays, but requires a somewhat larger amount of irradiation than necessary to destroy the infectivity of the suspensions. The effects of irradiation on the antiviral antibody present in the blood of animals which have become immune to the virus-an antibody that specifically fixes complement in mixture with the papilloma virus-are also conditioned by extraneous material. 250,000 to 500,000 r had only a slight effect on the antibody in whole serum, while this amount of irradiation completely inactivated comparable amounts of antibody in preparations partially purified by precipitation with ammonium sulfate. As a whole the findings indicate that under certain conditions of purity and concentration most of the radiation does not act by direct hits on virus or antibody particles, but indirectly by ionizing or exciting some other molecules present in the exposed suspension, which then react with the virus or antibody molecules.

THE DETECTION OF A "MASKED" VIRUS (THE SHOPE PAPILLOMA VIRUS) BY MEANS OF IMMUNIZATION

A study has been made of the immunization procedure described by Shope, with particular reference to the detection of "masked" papilloma virus by means of it. Papilloma extracts were frequently encountered which, though non-pathogenic, elicited the specific antiviral antibody and induced resistance to the virus upon injection intraperitoneally into normal rabbits. The results of the immunization experiments were often complicated, however, by the effects of extravasated antibody, which had accumulated in various amounts in many of the papillomas and was consequently present in extracts of them together with "masked" virus. The extravasated antibody was often sufficient to render extracts of domestic rabbit papillomas non-antigenic; and sometimes, when present in excess, its passive transfer conferred resistance to reinfection with the virus. The conclusion seems warranted that only positive immunization findings can be interpreted with certainty. Negative results provide no decisive evidence as to whether "masked" virus is or is not present in the injected material, unless the amount of extravasated antibody also present is known. The findings may have a bearing on the negative outcome of immunization experiments with extracts of the cancers deriving from the natural papillomas of cottontails. Crude suspensions of domestic rabbit papillomas, which contain little or no virus demonstrable by ordinary methods, are far less antigenic than extracts of the natural growths of wild rabbits, which contain virus in quantity. In explanation of the finding the possibility seems worthy of attention that domestic rabbit papillomas may contain much less virus than the growths of cotton tails, the natural hosts of the virus.

IMMUNOLOGICAL REACTIONS WITH A VIRUS CAUSING PAPILLOMAS IN RABBITS

The antigen that binds complement in the presence of sera neutralizing the Shope papilloma virus can be readily extracted from papillomas yielding infectious virus, but not from the normal skin of rabbits bearing the growths. The virus and the complement-binding antigen appear to have the same particle size, as determined by filtration, and they are thrown down together in the centrifuge. They are destroyed by the same amounts of heating and, in general, by the same changes in pH. It is possible, nevertheless, by irradiation with ultraviolet light, or by treatment with weak alkali, to render papilloma extracts non-pathogenic without diminishing their capacity to bind complement when mixed with immune serum.

Antigenicity and Infectivity of Extracts of Virus-Induced Rabbit Papillomas

The antigen which binds complement when mixed with sera neutralizing the Shope papilloma-virus bears an evident relation to the virus itself; for it is present in high titer in virus-containing extracts of papillomas, it is retained by filters which hold back the virus, and its effectiveness is destroyed by the same amount of heating which renders the virus non-pathogenic.1 The experiments now to be reported throw further light on this relation.Tests with Extracts of Non-Infectious Papillomas and of Normal Skin. On inoculation the Shope virus gives rise to papillomas in both domestic and cottontail rabbits; yet only infrequently can it be extracted in active state from the domestic-rabbit growths, and occasionally those of cottontails fail to yield it. Papillomas from 6 cottontail and 8 domestic rabbits, which were produced by the virus yet from which it could not be recovered, have been extracted as usual and tested repeatedly for capacity to bind complement under optimal conditions. Most of these non-in...