Comparative analysis of SV40 17kT and LT function in vivo demonstrates that LT's C-terminus re-programs hepatic gene expression and is necessary for tumorigenesis in the liver.

Transformation by Simian Virus 40 (SV40) large T antigen (LT) is mediated in large part by its interaction with a variety of cellular proteins at distinct binding domains within LT. While the interaction of LT's N-terminus with the tumor suppressor Rb is absolutely required for LT-dependent transformation, the requirement for the interaction of LT's C-terminus with p53 is less clear and cell- and context-dependent. Here, we report a line of transgenic mice expressing a doxycycline-inducible liver-specific viral transcript that produces abundant 17kT, a naturally occurring SV40 early product that is co-linear with LT for the first 131 amino acids and that binds to Rb, but not p53. Comparative analysis of livers of transgenic mice expressing either 17kT or full length LT demonstrates that 17kT stimulates cell proliferation and induces hepatic hyperplasia but is incapable of inducing hepatic dysplasia or promoting hepatocarcinogenesis. Gene expression profiling demonstrates that 17kT and LT invoke a set of shared molecular signatures consistent with the action of LT's N-terminus on Rb-E2F-mediated control of hepatocyte transcription. However, 17kT also induces a unique set of genes, many of which are known transcriptional targets of p53, while LT actively suppresses them. LT also uniquely deregulates the expression of a subset of genes within the imprinted network and rapidly re-programs hepatocyte gene expression to a more fetal-like state. Finally, we provide evidence that the LT/p53 complex provides a gain-of-function for LT-dependent transformation in the liver, and confirm the absolute requirement for LT's C-terminus for liver tumor development by demonstrating that phosphatase and tensin homolog (PTEN)-deficiency readily cooperates with LT, but not 17kT, for tumorigenesis. These results confirm independent and inter-dependent functions for LT's N- and C-terminus and emphasize differences in the requirements for LT's C-terminus in cell-type dependent transformation.

PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen.

Viruses of the DNA tumor virus family share the ability to transform vertebrate cells through the action of virus-encoded tumor antigens that interfere with normal cell physiology. They accomplish this very efficiently by inhibiting endogenous tumor suppressor proteins that control cell proliferation and apoptosis. Simian virus 40 (SV40) encodes two oncoproteins, large tumor antigen, which directly inhibits the tumor suppressors p53 and Rb, and small tumor antigen (ST), which interferes with serine/threonine protein phosphatase 2A (PP2A). We have constructed a Drosophila model for SV40 ST expression and show that ST induces supernumerary centrosomes, an activity we also demonstrate in human cells. In early Drosophila embryos, ST also caused increased microtubule stability, chromosome segregation errors, defective assembly of actin into cleavage furrows, cleavage failure, a rise in cyclin E levels and embryonic lethality. Using ST mutants and genetic interaction experiments between ST and PP2A subunit mutations, we show that all of these phenotypes are dependent on ST's interaction with PP2A. These analyses demonstrate the validity and utility of Drosophila as a model for viral oncoprotein function in vivo.

Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice.

Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.

Tissue-type plasminogen activator-induced invasion and metastasis of murine melanomas.

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.

E6 and E7 expression from the HPV 18 LCR: development of genital hyperplasia and neoplasia in transgenic mice.

Human papillomavirus type 18 infection is highly associated with malignant tumors of the genital tract. To investigate the tissue specificity of the HPV long control region (LCR) and the transforming ability of the E6-E7 oncoproteins, an HPV-18 transgene containing the viral LCR and E6 and E7 genes was introduced into mice. Three founder males exhibited enlarged seminal vesicles and preputial glands by 50 weeks of age. A line of transgenic mice was established by in vitro fertilization, and subsequent generations of transgenic males and females were monitored for lesions. Approximately 80% of hemizygous transgenic males exhibited enlarged seminal vesicles and preputial glands as early as 12 weeks of age. Histological examination indicated that this enlargement was due to distension by fluid, along with polyploid hyperplasia of the lining secretory epithelium. E6 and E7 transcripts were limited to affected organs and kidney. Approximately 41% of transgenic females developed cervical neoplasms between 1-2 years of age. Histologically, tumors were mesenchymal rather than epithelial in origin. E6 and E7 transcripts were restricted to cervical tumor tissue and kidney. These findings suggest that the HPV-18 LCR has an element(s) which directs expression specifically to the urogenital tract in transgenic mice.

Production of a phosphorylated GST::HPV-6 E7 fusion protein using a yeast expression vector and glutathione S-transferase fusions.

A Saccharomyces cerevisiae GAL7 expression vector for the production of protein fusions to glutathione S-transferase (GST) has been constructed. Using this vector, a GST fusion to human papillomavirus type 6 (HPV-6) E7 protein was produced and purified by affinity chromatography in a single step, at a yield of 2 micrograms/ml of culture. The E7 portion of the fusion protein was phosphorylated, in contrast to the same product made in Escherichia coli. Therefore, yeast GST vectors may be of specific use in producing phosphoproteins, or proteins with other eukaryotic post-translational modifications, in preparative amounts for in vitro analysis.

Characterization of intraocular tumors arising in transgenic mice.

PURPOSE: To characterize intraocular tumors that arise by in situ transformation in the choroid-retinal pigment epithelium (RPE) in transgenic mice bearing the SV40 oncogene under the control of the mouse tyrosinase promoter. METHODS: Tumors from TySV40 transgenic mice were characterized in vivo and in vitro by immunohistology, compound microscopy, and electron microscopy. Tumor cell lines were established and characterized for growth and metastatic potential in the eyes of nude mice. RESULTS: On light microscopy, ocular tumors were predominantly epithelioid, although occasional clusters of spindle cells were also present. Transmission electron microscopy revealed the presence of numerous basal infoldings and abundant multilaminated basement membranes on the ocular tumors. Tumors stained with antibodies to melanoma-associated antigens, gangliosides GD2 and GD3, and the SV40 T antigen. Radiolabeled transgenic tumor cells preferentially localized in the liver after intravenous injection in normal mice. Intracamerally transplanted transgenic tumors metastasized from the eyes to the livers of nude mice. CONCLUSIONS: In TySV40 transgenic mice, intraocular tumors develop that arise at the choroid-RPE interface, and they display morphologic and ultrastructural features consistent with RPE carcinomas. However, the transgenic tumors express melanoma-associated antigens and a propensity to metastasize to the liver, two features characteristic of uveal melanomas. The TySV40 transgenic murine tumors represent potentially useful tools for investigations into the biology and metastasis of intraocular neoplasms.

Rejection of intraocular tumors from transgenic mice by tumor-infiltrating lymphocytes.

The present study examined the role of tumor infiltrating lymphocytes (TIL) in the rejection of intraocular tumors from SV40 transgenic mice. Tumor cells from an intraocular tumor arising in an SV40 transgenic FVB/N mouse were transplanted into the eyes of syngeneic FVB/N mice and the TIL isolated. TIL were assessed for direct cytolytic activity in vitro. TIL were also transferred passively to immunosuppressed FVB/N mice to determine if they could mediate intraocular tumor rejection. The role of CD4+ and CD8+ T cells in intraocular tumor rejection was evaluated by depleting the respective cell populations in FVB/N hosts prior to intraocular tumor challenge. The results showed that intraocular tumors undergoing rejection in immunocompetent syngeneic hosts became infiltrated with T cells, with the CD8+ subset predominating at the time of rejection. By contrast, athymic nude mice did not reject the intraocular tumors nor did the tumors become infiltrated with TIL. TIL displayed direct, tumor-specific cytolytic activity immediately after isolation from the tumor-containing eyes. FVB/N hosts depleted of CD4+ T cells were unable to reject their intraocular tumors. In vivo depletion of CD8+ T cells delayed, but did not prevent tumor rejection. Adoptively transferred TIL mediated swift rejection of intraocular tumors in immunoincompetent recipients. Recipients of TIL, but not recipients of normal spleen cells, acquired significant tumor-specific CTL activity that was demonstrable in vitro. The results strongly suggest, but do not prove, that TIL mediate rejection of intraocular tumors from transgenic mice by direct cytolysis. Although CD4+ T cells are necessary for tumor rejection and are capable of direct cytolysis, the predominant effector cells are CD8+ CTL.

T cell responses to the human papillomavirus type 16 E7 protein in mice of different haplotypes.

The response of murine T cells to the E7 molecule of human papillomavirus type 16 (HPV-16) was studied using eight different mouse strains of six distinct H-2 haplotypes. HPV-16 E7 protein was prepared as a fusion protein with glutathione-S-transferase, purified by affinity chromatography and used for immunization. Cells from the lymph nodes were cultured with whole fusion protein, glutathione-S-transferase or HPV-16 E7 protein synthetic peptides. All the mouse strains tested, with the exception of BALB/c, recognized the E7 molecule, as evidenced by a proliferative response to at least two of the peptides. The profile of responses to peptides varied between and within a strain, but five distinct immunodominant regions could be identified. These regions were defined on the basis of a reaction to one or more peptides in a given part of the E7 molecule by at least four strains. The five regions were encompassed by amino acid residues 1 to 9, 17 to 32, 42 to 59, 62 to 77 and 87 to 98. The findings suggest that in an outbred population, such as man, the E7 molecule of HPV-16 would be recognized by a large proportion of the population. However, the poor response of two mouse strains [B10.RIII (71NS) and BALB/c] could also have a corollary in man.

Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant.

Leishmania (Viannia) braziliensis: comparative pathology of golden hamsters infected with isolates from cutaneous and mucosal lesions of patients residing in Tres Bracos, Bahia, Brazil.

The histopathology of primary forepaw and metastatic lymph node, spleen, and liver lesions produced in golden hamsters infected with cutaneous leishmaniasis (CL) strains (LTB 111 and LTB558) and mucocutaneous leishmaniasis (MCL) strains (LTB12 and LTB201) of Leishmania (Viannia) braziliensis isolated from patients residing in Tres Bracos, Bahia, Brazil is described. No pathological features providing clear differentiation of the CL and MCL strains were found. Although amastigotes were plentiful early in the development of primary forepaw lesions, they were either absent or could not be identified with certainty in sections of late stage lesions. Similarly, amastigotes were not found in histologic lesions at metastatic sites; however, leishmanial DNA was detected in both early and late stage forepaw lesions and metastatic lesions using Leishmania kinetoplast DNA and the gene coding for gp63 as hybridization probes. The DNA recovered from metastatic lesions was extracted from formalin-fixed paraffin-embedded tissues that had been stored at room temperature for prolonged periods.

The isolation of nuclear envelopes. Effects of thiol-group oxidation and of calcium ions.

The effects of (a) oxidative cross-linking of protein thiol groups and (b) the presence or absence of Ca2+ ions on rat liver nuclear-envelope isolation were studied. Two envelope-isolation procedures were compared: a well characterized low-ionic-strength method and a recently developed high-ionic-strength method. The latter method seems preferable to the former in respect of lower intranuclear contamination of the envelopes, suppression of endogenous serine proteinase, and maintenance of high specific activities of envelope-associated enzymes. In both procedures, however, the presence of Ca2+ gave rise to a rapid, apparently irreversible, contamination of the envelopes by intranuclear material. This effect was half-maximal at 20 microM-Ca2+. In addition, the envelopes became contaminated with intranuclear material by a Ca2+-independent mechanism, apparently resulting from N-ethylmaleimide-sensitive intermolecular disulphide-bond formation. This oxidative process seemed to have two major kinetic components (half-life, t1/2, approx. 2 min and 10 min). In view of these findings, it is recommended that (i) for most purposes, nuclear envelopes be isolated by the newly developed high-ionic-strength procedure, (ii) irrespective of the method used, Ca2+-chelators be included in all the buffers, (iii) thiol-group oxidation be prevented or reversed during the procedure.