Uropod-bearing lymphocytes (hand mirror cells) in a virus-induced murine lymphoma.

A Moloney murine leukemia virus-induced T-cell preleukemic thymic lymphoma tissue culture from an inbred C3H/HeJ mouse contained numerous hand mirror cells (HMC). The cells were studied by light and phase-contrast microscopy, special stains, indirect immunofluorescence for terminal deoxynucleotidyl transferase, and scanning and transmission electron microscopy. The uropods of the mouse and human HMC were similar. In contrast, viruses were noted on the tip of the mouse HMC uropod by transmission electron microscopy. These observations, reported for the first time in an animal model, will enable investigators to study the HMC under controlled conditions.

Electron microscopic studies of intracisternal virus particles in Soehner-Dmochowski murine sarcoma virus-induced bone tumors of New Zealand Black rats.

Soehner-Dmochowski murine sarcoma virus (Moloney)-induced bone tumors of New Zealand Black rats carry two morphologically different types of virus particles, namely, extracellular type C and intracisternal virus particles, which have thus far not been reported. These two types of virus particles have also been observed in the tissue culture cells derived from normal prostate tissues of A/Dm and BALB/c/Dm mice after inoculation of cell-free extracts of these bone tumors. The intracisternal virus particles, 90 to 120 nm in diameter, have always been found in the rough endoplasmic reticulum; they have two inner concentric layers with a relatively electron-lucent center, frequently showing cylindrical, chain-like, or multipolar budding forms. Type C virus particles produced by Soehner-Dmochowski murine sarcoma virus (Moloney)-infected prostate tissue culture cells from A/Dm and BALB/c/Dm mice belong to the murine sarcoma-murine leukemia virus group, as revealed by the fixed immunofluorescence test and by immunoelectron microscopy. The morphological and immunological relationship of intracisternal virus particles and other types of virus particles (such as type C, type H, and intracisternal type A virus particles) and intracisternal virus particles in guinea pig leukemia are defined by routine electron microscopy observations and by immunoelectron microscopy studies.

Induction of sister chromatid exchange by polyoma large viral tumor antigen in transformed rat fibroblasts.

The frequency of sister chromatid exchange (SCE) was determined in rat fibroblasts transformed by wild-type polyoma virus or by a mutant temperature sensitive for viral large tumor antigen function (ts-a). Elevated SCE frequencies were observed in two wild-type transformed cell lines growing at 37 degrees and in four ts-a-transformed lines upon growth at the permissive temperature for large viral tumor antigen (33 degrees). The increase in SCE frequency in ts-a-transformed cells at 33 degrees was reversed by growth at 39 degrees (nonpermissive for T-antigen function). An increase in SCE at 33 degrees was not observed in untransformed cells or in a ts-a-transformed cell line which makes a defective large viral tumor antigen. These results suggest that large viral tumor antigen can induce SCEs. Since large viral tumor antigen is also responsible for amplification of integrated viral DNA sequences (4), we tried to correlate this phenomenon with the increased SCE frequency. However, increasing SCE artificially by growing cells in the presence of 12-O-tetradecanoylphorbol-13-acetate did not result in amplification of integrated viral DNA in the absence of large viral tumor antigen function. Thus, there is no simple causal relationship between increased SCE and amplification.

Mammary tumor virus DNA as a marker for genotypic variance within hormone-responsive GR mouse mammary tumors.

Mammary tumors of GR mice acquire extra mammary tumor virus (MMTV) DNA information within their DNA during tumor growth and development. These extra MMTV genes have been used by us as genotypic markers to investigate the heterogeneity of GR mammary tumors and their loss of hormone dependence during serial transplantation. Our studies reveal that the various subpopulations of cells within individual GR mammary tumors are characterized by differences in number and location of acquired extra MMTV DNA fragments. Losses of certain of these extra MMTV DNA fragments occur when mammary tumors become hormone independent, indicating a loss of hormone-dependent cells. The study of MMTV DNA markers also reveals that low levels of autonomous cells are already present in some hormone-dependent mammary tumors at an early stage of their development. The genotypic analysis strongly indicates that mammary tumor progression is not due to phenotypic adaptation but to clonal selection of the more aggressive sublines.

Correlation between the loss of the transformed phenotype and an increase in superoxide dismutase activity in a revertant subclone of sarcoma virus-infected mammalian cells.

We have studied the effects of paraquat (methyl viologen), a herbicide that increases intracellular production of superoxide radical, on the viability of virus-transformed and nontransformed normal rat kidney (NRK) cells in culture. We have shown that a low concentration of paraquat (12.5 microM) is cytotoxic toward virus-transformed cell lines, including Kirsten sarcoma virus- and SV40-transformed NRK cells. The corresponding untransformed NRK cells were resistant to the same and a 4-fold higher concentration of paraquat. There was a good correlation between the susceptibility of transformed and untransformed cells to paraquat cytotoxicity and their ability to increase the superoxide dismutase (SOD) enzymatic activity. We found that paraquat is cytotoxic toward Kirsten sarcoma virus-transformed and SV40-transformed NRK cells which showed low intracellular SOD activity. The relationship between SOD activity and paraquat cytoxicity was strengthened by the finding that the tolerance of NRK cells to the drug was associated with high intracellular SOD activity. This report also describes the isolation of a revertant (revertant RE8G3) cell line derived from Kirsten sarcoma virus-transformed NRK cells after paraquat treatment which contains SOD activity at levels much higher than those found in NRK cells. This revertant is undistinguishable from NRK cells with respect to its lack of transformed cell properties. Not only are these cells normal morphologically but also they do not grow in soft agar, an in vitro property that closely correlates with in vivo tumorigenicity. Several biological and biochemical properties of RE8G3 cells, including growth characteristics, surface receptors for both transferrin and epidermal growth factor (EGF), and the EGF-dependent 32P phosphorylation of specific membrane polypeptides have been studied. The most interesting conclusion that can be drawn from these studies is that there is a correlation between loss of the transformed phenotype and an increase in both EGF receptors and EGF-dependent 32P phosphorylation of a m.w. 170,000 membrane-associated protein.

SV-40 virus-induced neoplastic transformation of hamster brain cells in vitro. Studies with scanning and transmission electron microscopy.

Neoplastic transformation of one-day-old hamster brain cells was produced by infection with SV-40 virus and verified by phase contrast microscopy, growth in semisolid media, and intracranial tumor production after inoculation of the cells into other one-day-old hamsters. Transformed cells were studied by transmission and scanning electron microscopy. The numerous alterations in cell surface structure and in nuclear and cytoplasmic organization suggest a marked increase in cell metabolism and in the rate of mitosis and cell division. Cilia with a nine-to-zero pattern of microtubule doublets were present in cells with intermediate size filaments which stained for glial fibrillary acidic protein. The findings indicate that infection of one-day-old hamster brain cells in culture by SV-40 virus results in their transformation to a neoplastic state and the transformed cells are differentiating neoplastic astrocytes.

Human papillomavirus (HPV) in condylomatous lesions of cervix.

Ninety-seven cervical condylomata classified histologically as flat condyloma (planum), papillary condyloma (acuminatum), and endophytic condyloma were studied by transmission electron microscopy (TEM) and by immunoperoxidase technique (IPT) for the presence of human papillomavirus (HPV) particles and antigen, respectively. Both techniques localized HPV chiefly in nuclei of koilocytotic cells. HPV particles were found in 25% of the cases by TEM and HPV antigen was detected in 48% of the cases by IPT. All cases positive by TEM were also positive by IPT, thus confirming the specificity of the immunological staining. The viral antigen was detected in 56% of 68 flat condylomata, 35% of 26 papillary condylomata, and in none of 3 cases of endophytic condylomata. However, when histiotypes of virus-positive condylomata were controlled for the intraepithelial extent of koilcytotic cells, the prevalence of HPV correlated with the extent of koilocytosis rather than with the histiotype. The immunologic technique will be of value for the further characterization of cervical condylomata and of the relationship between HPV infection and cervical intraepithelial neoplasia.

Feline uveal melanoma model induced with feline sarcoma virus.

This paper described the first animal model of a virally induced uveal melanoma. Tumors developed following the injection of an RNA tumor virus, i.e., Gardner strain feline sarcoma virus, into the anterior chamber of newborn kittens. Histologically, the tumors were found to be iris and ciliary body melanomas, many of which showed invasion. The histology and ultrastructure of those tumors are described.

Human papillomavirus lesions in association with cervical dysplasias and neoplasias.

A series of 620 cervical biopsy specimens (precancerous and malignant) was assessed morphologically with special reference to the concomitant appearance of human papillomavirus lesions. Tissue samples from 346 of these biopsy specimens were stained for human papillomavirus antigens using the immunoperoxidase-peroxidase-anti-peroxidase (PAP) technique. Papillomavirus lesions were found in 55.6% of the biopsy specimens associated with all degrees of epithelial atypia. The mean age of the women with papillomavirus (condylomatous) changes was significantly lower (P less than .0001) than that of women without these lesions, ie, those who had dysplasia/neoplasia without concomitant papillomavirus changes. Flat and inverted condylomas were most frequent between the ages 20 and 39 and were accompanied by more severe dysplasias than the papillomatous condylomas. In immunoperoxidase-PAP staining, 56% of the papillomavirus lesions were positive, the positivity being inversely related to the degree of epithelial atypia, and bearing some correlation with the condyloma type (papillary 100%, inverted 70%, and flat 52%). Although the results show a clear-cut association of human papillomavirus lesions with premalignant, and to a lesser extent with malignant squamous cell lesions of young sexually active women, thus suggesting a relationship between the virus and cancer, a careful follow-up study is needed to fully elucidate this relationship.

Epidermodysplasia verruciformis. Clinical and light- and electron-microscopic observations during etretinate therapy.

Three patients with epidermodysplasia verruciformis (EV) were treated with etretinate for 9-13 months. The patients had lesions characteristic of EV, including flat warts, common genital warts, pityriasis-versicolor-like lesions and malignant changes such as actinic keratosis and Bowenoid cancer in situ. During etretinate treatment, some flattening of the warts was observed in all three patients, and the lesions on the chest and back became less red and scaling. However, none of the lesions disappeared completely, and when the treatment was discontinued, the lesions relapsed. No malignant changes were detected during the period of therapy. Electron microscopy revealed the presence of typical large, clear cells containing viral particles in the upper epidermis. Etretinate therapy induced the same type of fine-structural changes as those seen in keratinization disorders and genodermatoses. The clear cells and virus particles persisted throughout the treatment period. More long-term, controlled studies are necessary to make possible an estimate of the curative and cancer-inhibitory effect of etretinate treatment in patients with EV.

[Viral acanthomas and specialized forms of keratinosome "membrane coating granules" (author's transl)]. / Virusacanthome und besondere Formen von Keratinosomen "Membrane Coating Granules".

In the case of viral acanthomas, the stratum spinosum and granulosum presents ballooned cells which contain all transitional stages from multivesicular bodies (MVB) to keratinosomes. A particularity in condylomata acuminata are the "wagon-wheel" bodies. These structures are typical for the non keratinazed squamous epithelium. The participation of intercellular extruded "wagon-wheel" bodies, MVB and atypical keratinosomes on an irregular baso-apical diffusion-barrier in the epidermis of cases with viral acanthomas has been discussed. On the basis of the relation seen between MVB and the Golgi-apparatus, their transition to partially atypical keratinosomes in cases of viral acanthomas and their "expulsion" into the intercellular space could indicate that in keratinozytes the enzymatically regulated feed-back between the cellular surface and the capability to synthesize is changed by viral agents. The interference appears to manifest itself in the Golgi-apparatus and also appears to be "specified" by the terrain present.

Association of HPV with human genital tumors.

Human papillomaviruses (HPV) are clearly responsible for the induction of genital lesions like condylomata acuminata, bowenoid papules, and flat condylomas. Moreover, the DNA of particular virus types (HPV 16 and 18) is found in a substantial number of invasively growing squamous cell carcinomas of the genital tract, suggesting an etiologic involvement of these viruses in tumor development. Since HPV 16 and 18 as well as other papillomaviruses (HPV 6 or 11) usually present within the benign genital warts can be found in dysplastic lesions of the uterine cervix known as putative precancerous lesions, determination of the virus type might be of diagnostic relevance. Since no type-specific serologic reagents are available, viruses can be identified by nucleic acid hybridization using radioactively labeled HPV DNAs that have been molecularly cloned as probes.

Electron microscopy in assessment of the biological behavior of human papillomavirus infections in the uterine cervix.

To asses the natural history of human papillomavirus (HPV) infections in uterine cervix, currently implicated in etiology of cervical cancer, a prospective follow-up study has been conducted for 418 women at our clinic since 1981. The present communication summarized the current follow-up data of these patients, with special emphasis on detection of the virus in cervical punch biopsies, as correlated with other characteristics pertinent to the clinical behavior of cervical HPV infections. On each attendance, the patients are subjected to colposcopy accompanied either by Papanicolaou (PAP) smears or punch biopsies. The latter are analyzed for the cytopathic changes of HPV, for concomitant cervical intraepithelial neoplasia (CIN), for HPV structural proteins with IP-PAP technique as well as on transmission electron microscopy (TEM) for the presence of HPV particles. The local immunocompetent cell (ICC) infiltrates are analyzed using ANAE technique to define B cells, MPS cells and T cells and monoclonal antibodies (McAb) for T cell subsets, NK (natural killer) cells and Langerhans cells. HPV particles were disclosed with equal frequency (approx. 65%) in all three types of HPV lesions. Surprisingly, HPV particles were present in 70% of the biopsies derived from the regressed lesions (e. g. in those without histological evidence of HPV lesions), suggesting a possibility of a latent HPV infection. Presence of viral particles did not bear any direct correlations with the expression of HPV antigens, intensity or cellular composition of the ICC infiltrate, defined by ANAE or using McAb. Presence of HPV particles was not a major prognostic determinant, whereas the clinical course was most significantly influenced by the grade of HPV-associated CIN, to which regression was inversely and progression directly related. The results clearly confirm that cervical HPV infections are capable of progressing into carcinoma in situ and thus present with a natural history equivalent to that of classical CIN.

Isolation and partial characterization of plasma membranes from chicken liver and from Mc-29 virus induced transplantable hepatoma.

Plasma membranes have been isolated from chicken liver and from Mc-29 virus induced transplantable hepatoma. The purity of membrane preparations has been checked by electron microscopy and by determination of the activity of some enzymes: 5'-nucleotidase, Na+, K+-ATP-ase, Mg2+-ATP-ase, alkaline beta-glycerophosphatase and glucose-6-phosphatase. In hepatoma membranes the activity of 5'-nucleotidase, Na+, K+-ATP-ase and Mg2+-ATP-ase was lower, that of alkaline phosphatase higher, than in liver membrane preparation. The incorporation rate of glucosamine-14C into UDP-N-acetylglucosamine and into plasma membrane glucosamine have been studied as well. The rate of synthesis of UDP-N-acetylglucosamine was faster in liver than in tumor cells. The labeling of hepatoma plasma membranes with glucosamine-14C occurred more slowly than that of liver ones. The rate of transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to membrane-bound glucosamine is lower in hepatoma, than in liver cells.

Electron microscopic features of a brain tumor induced in hamster by BK virus, a human papova virus.

In order to locate the target cells for malignant transformation by BK virus (a human papova virus) in hamster brain, electron microscopic observation of tumor originally induced in hamster brain by BK virus was performed. With light microscopy, the BK virus-induced tumor (Vn 17) bore a close resemblance to human malignant ependymoma. Under the electron microscope, numerous microvilli and few cilia were visible on the surface of the tumor cells. These tumor cells were joined to each other by desmosomes. Gap junctions were not observed. Multilayered cuboidal cells were observed around the lumen and blood vessels in the tumor. With regard to fine structure, three types of Vn 17 cells were recognized; ependymal like cells, tanycytes with prominent cell processes, and undifferentiated cells with few cytoplasmic organelles. There was no basal lamina between the ependymal cells and the connective tissue stroma. The Vn 17 cells showed some similarity to the ultrastructural features of the epemdymal cells of newborn rabbits, suggesting that the target cells for Vn 17 may be cells related to ependyma. Malignant transformation of the cells would be initiated in the early stages after BK virus inoculation into the brain of newborn hamsters.

Laryngeal papilloma cells in culture have an altered cytoskeleton.

We have investigated the effect of human papillomavirus (HPV) transformation on cellular cytoarchitecture. Cells from laryngeal papillomas and normal epithelium were cultured in vitro. Cytoskeletal components of both types of cells were visualized by immunofluorescence, to determine whether there were any differences in the structure or distribution of the cytoskeleton. There was no significant change in microtubules. Two major components of the cytoskeleton, the intermediate filaments and the microfilaments, were altered in the papilloma cells. Polyacrylamide gel electrophoresis of isolated keratins showed differences between normal and papilloma tissue, which might explain the altered intermediate filament distribution. The changes in cytoskeletal structure may be one way in which HPVs alter cellular growth controls.

Papilloma-like virus infection in Bolivian side-neck turtles.

Five Bolivian side-neck turtles had multifocal small, round to confluent, white skin lesions distributed over the head. Several gram-negative microorganisms were isolated from the lesions. Light microscopy revealed hyperkeratosis and hyperplasia of the epidermis. Ultrastructural evaluation demonstrated crystalline aggregates of virus particles within nuclei of cells in the stratum granulosum and free within the stratum corneum. On the basis of size, location, arrangement, and tissue affected, the particles resembled papillomaviruses.

Immunofluorescence and electron microscopic investigations of epithelial hybrid cells derived from nasopharyngeal carcinoma (NPC-KT).

The present study was made to investigate some characteristics of the epithelial hybrid cells derived from nasopharyngeal carcinoma (NPC-KT cells) both in vivo and in vitro, using immunofluorescence and electron microscopic techniques. Immunofluorescence and electron microscopic studies have shown that the appearance of Epstein-Barr virus (EBV)-related early antigens, EB-viral capsid antigens and virus particles in nude-mouse-grown-tumour cells were rather repressed, in contrast to, in vitro culture of the NPC-KT cells. The tumours after transplantation of the NPC-KT cells to nude mice showed pathological pictures of poorly differentiated carcinoma with EBV-associated nuclear antigen and derived from the NPC-KT cells by means of cytogenetic studies. More importantly, we have detected EBV-related membrane antigens (MA) on the epithelial NPC-KT cells. To our knowledge, the presence of MA on the malignant epithelial cells of the nasopharynx have never been demonstrated. The results reported here show for the first time the presence of MA on nasopharyngeal carcinoma cells.