Regression,proliferation and development of lip papillomas in wild white suckers,Catostomus commersoni,held in the laboratory

Synopsis Epizootics of skin neoplasms in teleost species have been documented within the Great Lakes region over the last decade. The white sucker,Catostomus commersoni, has been proposed as a sentinel species to monitor environmental health in these systems. The prevalence of skin neoplasia is elevated in white suckers and other fish taken from chemically polluted sites or from lake regions adjacent to heavy industry. Lip papillomas regress and proliferate spontaneously in captive wild suckers. It is important to investigate the relevance of these observations to papilloma etiology to determine whether the prevalence of this disease is a suitable biomarker for environmental health. White suckers were captured in the spring of 1992 and 1993 during annual spawning runs (mid to late April) at the Ganaraska River, which discharges into Lake Ontario at Port Hope, Ontario. Under crowded laboratory conditions, there was either proliferation of existing papillomas or development of new papillomas. However, in uncrowded conditions, existing papillomas either regressed completely or there was no development of new papillomas. Protein Kinase C (PKC), a proposed marker enzyme for hyperplasia and neoplasia, was used to determine if regressing and proliferating papillomas could be differentiated on the basis of biochemical activity. PKC activity was lower in proliferating papillomas, but not significantly different from papillomas sampled initially from Ganaraska River suckers. Regressing papillomatous tissue displayed a significantly higher level of enzyme activity than either proliferating or unchanged papillomas, but the PKC activity of regressing papillomas was not significantly different from that of normal lip epidermis.     

Expression of retinoblastoma gene product in respiratory epithelium and sinonasal neoplasms: relationship with p16 and cyclin D1 expression

Transition from G1 to S phase of the cell cycle is mediated by interactions between the Retinoblastoma gene product (pRb), p16, and cyclin D1. To determine the expression of these proteins in the sinonasal mucosa immunohistochemistry was carried out on archived tissue sections from 46 patients (37 men, 9 women, age range 17 to 82 years, median 55 years). Nuclear immunostaining for these proteins was assessed and the expression rates (percentages of immunoreactive nuclei) in normal respiratory epithelium, inverted sinonasal papillomas, cylindrical (oncocytic) sinonasal papillomas, and squamous cell carcinomas were compared. Normal respiratory epithelium showed significantly higher pRb expression in surface cells compared to basal cells (p < 0.05). In contrast, abundant pRb expression in surface and basal cells was detected in columnar differentiation in sinonasal papillomas and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed significantly reduced pRb expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05, respectively). Expression of p16 was detected in all epithelial cell layers of normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed increased p16 expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05 between squamous metaplasia and columnar epithelium). Sinonasal squamous cell carcinomas showed the coexpression of pRb and p16. Expression rates of cyclin D1 higher than 10% were detected only in invasive carcinomas but not in carcinoma in situ, sinonasal papillomas or respiratory epithelium. Conclusively, pRb expression accompanies terminal differentiation in columnar surface cells. Expression of pRb in proliferating basal cells is present in sinonasal papillomas and adjacent mucosa but not in normal respiratory epithelium. Cuboidal and squamous metaplasia in inverted papillomas involves downregulation of pRb expression along with increased p16 expression in surface cells. Sinonasal squamous cell carcinomas coexpress pRb and p16. Overexpression of cyclin D1 in sinonasal lesions is confined to invasive squamous cell carcinomas.     

Detection of human papillomavirus in papillomas of oral cavity

The oral cavity is exposed to many harmful factors among others: tobacco, alcohol and viral infection e.g. human papillomaviruses (HPV). HPV belongs to a family of tumorigenic viruses and induces cutaneous and mucosal proliferations of epithelial cells. The aim of our study was to estimate the frequency of HPV occurrence in papillomas of oral cavity in the Podlasie region. The study included 38 papillomas of oral cavity. HPV was found in 14 cases (36.8%). High risk HPV was present in 12 papillomas, while low risk HPV was detected in 2 papillomas.     

Phenotypical characterization of lymphocytes infiltrating regressing papillomas.

Papillomavirus-induced lesions often regress spontaneously in both humans and animals. Papilloma regression is deemed to be due to a cell-mediated immune response, the nature of which is still ill defined, and is accompanied by immune cell infiltrates. To gain further information on the nature and role of the immune cells present in regressing papillomas, we have analyzed biopsies of papillomas induced in the soft palate of cattle by bovine papillomavirus type 4 (BPV-4) and have phenotypically characterized and quantified the lymphocytes present in these lesions. Eleven papilloma biopsies and seven biopsies of noninfected palate were analyzed for the presence of activated CD4+, CD8+, and gamma delta(WC1+) lymphocytes. We found large numbers of lymphocytes in the subepithelial derma of papillomas but not in normal palate tissue; these cellular masses consisted predominantly of CD4+ lymphocytes, with only a few CD8+ and gamma delta(WC1+) lymphocytes, generally positioned at the periphery of these masses. All three subtypes of lymphocytes were found interdigitated with the cells of the basal layer both in papillomas and in normal palate tissue, but while basal layer CD8+ and gamma delta(WC1+) T cells were detected with similar frequencies in papillomas and uninfected palate, basal layer CD4+ T cells were much more frequent in papillomas. CD4+, CD8+, and gamma delta(WC1+) lymphocytes were found in the suprabasal layers of papillomas, but the CD8+ and gamma delta(WC1+) T cells were more numerous and had migrated further into the differentiating keratinocytes of the papilloma fronds than the CD4+ T cells. We conclude that T-cell infiltration is characteristic of regressing BPV-4 papillomas, that CD4+ lymphocytes are specifically and massively recruited into the regressing papillomas, and that although all three lymphocyte subsets can penetrate the papilloma, only the CD8+ and gamma delta(WC1+) lymphocytes are able to migrate into the fronds. These results suggest that all three lymphocyte subsets have an important role to fulfill during natural regression of papillomas.     

Temperature-induced alterations in protein composition of newt papilloma cells

Protein patterns of Japanese newt papilloma in vivo at low (4 degrees C), normal (10 degrees C, control) and elevated (30 degrees C) temperature were investigated by two-dimensional gel electrophoresis. There were nine protein spots in normal skin (skin specific spots: SSS) which did not exist in papillomas. At 10 degrees C, the papillomas possessed three specific protein spots (papilloma specific spots: PSS) which did not appear in normal skin. At the reduced environmental temperature, eight of the nine missing proteins in papillomas had reappeared by 12 weeks exposure. Differential responses in reappearance of SSS in papillomas varied with environmental temperature. The PSS generally were unchanged by environmental temperature modulation, although one specific protein disappeared at 12 weeks at 4 degrees C. Reappearance of normal SSS in papillomas occurred early in treatment and reflected only minor variations in high versus low temperature exposures. These data suggest that temperature-induced tumor regression may be associated with changes in protein composition.     

Vaccination of rabbits with an adenovirus vector expressing the papillomavirus E2 protein leads to clearance of papillomas and infection

Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.     

Electron microscopy and immunostaining of the normal breast and its benign lesions. A search for neuroendocrine cells

Specimens from 7 patients with normal breast tissue 26 patients with benign breast lesions (6 fibroadenomas, and 4 intraductal papillomas, 2 mammae lactantes, 10 cases of cystic disease and 4 fibrotic lesions) were studied by immunocytochemistry and electron microscopy. Excretory epithelial cells in 2 of the 4 papillomas were immunostained for NSE. Myoepithelial cells were frequently stained as well. All the breast specimens were nonreactive to the antichromogranin antibody we used. The 2 NSE positive intraductal papillomas were tested for presence of hormone immunoreactivity, but no positively stained cells were observed. No cells with neuroendocrine features were observed by electron microscopy. The present study did not reveal neuroendocrine cells in the normal breast specimens and undisputed proof of neuroendocrine differentiation in benign breast lesions was not established. We conclude that if neuroendocrine cells are present in the normal breast, they are very rare, and probably not the cellular origin of all breast carcinomas with neuroendocrine features.     

p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).     

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.     

Transfection of the EJ rasHa gene into keratinocytes derived from carcinogen-induced mouse papillomas causes malignant progression.

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.     

Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms.

In an initial efforts to characterize the virological basis of neoplasia in the Shope papilloma-carcinoma system, the extent to which the viral genome is present in non-virus-producing benign and malignant tumors in domestic rabbits was established. Employing nick-translated radioactive viral DNA purified from productively infected papillomas on cotton tail rabbits as a probe, it was found that (i) papillomas, primary carcinomas, and metastatic carcinomas contain 10 to about 100 copies of the viral genome per diploid cell equivalent of DNA and (ii) viral DNA is present in detectable amounts in essentially all neoplastic cells. These results are consistent with the suggestion that continued presence of the viral genome is necessary for induction and maintenance of malignant as well as benign neoplasms.     

PCNA and P53 expression in relation to clinicopathological features of oral papilloma

Although papilloma is the most frequent benign epithelial tumour of oral cavity, its biological potential for malignant transformation is still to be evaluated. The aim of the study was to correlate PCNA and P53 expression in 55 oral papillomas with some clinicopathological variables. The tissue samples were stained with H+E and by immunohistochemistry for PCNA and P53 protein. Staining patterns were assessed semiquantitatively and correlated with each other and grade of tumour epithelial dysplasia, tumour size, localization well patient age and sex. PCNA immunostaining was positive 43 (78%) oral papillomas. P53 immunohistochemical reaction was positive in 38 (69%) out of 55 epithelial tumours. Positive relationship between PCNA and P53 expression was observed as well as between PCNA immunostaining and grade of epithelial dysplasia. There was no statistically significant relationships between PCNA, P53 immunohistochemical positivity and papilloma size, site, patient age and sex. The results of this study suggest that immunohistochemical P53 overexpression is valuable marker of early neoplastic transformation and together with PCNA are presumed predictors for malignant transformation of oral papillomas.     

Effect of vitamin A acid on papillomas induced by irradiated bed bugs in rabbit skin

The preventive effect of vitamin A acid (13-cis-retinoic acid) on skin papillomas induced in rabbits (Oryctolagus cuniculus) by the bite of bed bugs (Cimex lectularius) pre-irradiated with gamma rays was investigated. Painting the papillomas with an oily 13-cis-retinoic acid suspension twice a week in a dose of 25 mg/kg body weight leads to significant regression of these structures.     

The putative E5 open reading frame of cottontail rabbit papillomavirus is dispensable for papilloma formation in domestic rabbits.

In the cottontail rabbit papillomavirus (CRPV)-rabbit system, recombinant CRPV DNA can induce papillomas. This investigation was undertaken to evaluate whether the E5 open reading frame (ORF) of CRPV is required for papilloma formation. The CRPV genome we utilized, CRPV-WA, was sequenced in the E5 region and was found to contain one deletion, two insertions, and one transition mutation compared with CRPV-KS, the CRPV genome that has been fully sequenced. Despite these differences, an intact E5 ORF is preserved, supporting the notion that this gene may serve a biological function. One frameshift and two in-frame mutations were constructed in the small region of the 5' end of the E5 ORF that follows the E2 stop codon and precedes the L2 ORF. Several hundred rabbit skin sites were inoculated with each DNA preparation with a jet injector to test the ability of three CRPV E5 mutant DNAs to induce papillomas. In vivo results showed that each of the mutants induced papillomas, and biochemical analysis demonstrated that the E5 mutations present in DNA inocula were retained in the papillomas. The frequency of papilloma formation, however, was generally lower with each of the CRPV E5 mutants than with wild-type CRPV DNA, particularly so for the E5 frameshift mutant, suggesting that although the recognized E5 ORF is not required in domestic rabbits for the induction of papillomas by CRPV DNA, it may facilitate their formation.     

v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis

Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.     

Possible prognostic significance of p53 and Ki 67 in inverted sinonasal papilloma

The aim of this study is to test the possible prognostic significance of p53 and Ki67 expression in inverted papilloma of the lateral nasal wall and adjacent sinuses regarding their malignant potential and recurrence. 49 biopsies of the lateral nasal wall and adjacent sinuses obtained from 41 patients from three hospitals were investigated. Immunohistochemically demonstrated p53 and Ki67 expression was measured and statistically evaluated. p53 immunoreactivity was demonstrated in most of papillomas with carcinomas but only in two benign papillomas, while Ki67 demonstrated stronger immunoreactivity in carcinomas and surrounding epithelium. Immunohistochemical staining of inverted sinonasal papillomas for p53 and Ki67 can give useful information concerning the existence of synchronous carcinoma and, in case of high Ki67, a hint toward possible recurrence.     

Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.

The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.