Variant morphology and random chromosomal integration of BK polyomavirus in posttransplant urothelial carcinomas.

BK polyomavirus (BKPyV) causes major complications in solid organ transplant recipients but little is known about its role in the development of urothelial carcinoma (UC) during immunosuppression. Immunohistochemistry (IHC) screening for polyomavirus large T antigen (LTag) was performed in 94 micropapillary UC (MPUC), 480 unselected UC, 199 muscle invasive UC (including 83 UC with variant differentiation), 76 cases of plasmocytoid, nested and large nested UC and 15 posttransplant UC. LTag expressing UC were reevaluated regarding their histomorphological features and characterized by IHC for p53 and HER2, chromogenic in situ hybridization for HER2 and SNaPshot analysis of the TERT promoter and HRAS. Real-time PCR and next generation sequencing (NGS) were performed to search for BKPyV-DNA and for variants in the tumor and viral genomes. We detected five LTag expressing UC which were diagnosed between 2 and 18 years after kidney (n = 4) or heart (n = 1) transplantation. 89 MPUC without history of organ transplantation and overall 755 UC (including cases with variant histology) were LTag negative. Of the five LTag expressing UC, three were MPUC, one showed extensive divergent differentiation with Mullerian type clear cell carcinoma, and one displayed focal villoglandular differentiation. All five tumors had aberrant nuclear p53 expression, 2/5 were HER2-amplified, and 3/5 had TERT promoter mutations. Within the 50 most common cancer related genes altered in UC we detected very few alterations and no TP53 mutations. BKPyV-DNA was present in 5/5 UC, chromosomal integration of the BKPyV genome was detectable in 4/5 UC. Two UC with BKPyV integration showed small deletions in the BKPyV noncoding control region (NCCR). The only UC without detectable BKPyV integration had a high viral load of human herpesvirus 6 (HHV-6). Our results suggest that LTag expression of integrated BKPyV genomes and resulting p53 inactivation lead to aggressive high-grade UC with unusual, often micropapillary morphology.

Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses.

BACKGROUND: Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. OBJECTIVE: We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. METHODS: We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. RESULTS: We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification of intact virions by both electron microscopy and next-generation sequencing support a role for active viral infections in these skin diseases. LIMITATION: This was a small case series of archived materials. CONCLUSION: We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a distinctive histologic pattern and describe this entity as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses."

Presence of Merkel cell polyomavirus DNA and large-T antigen in keratinocyte carcinomas and its correlation with immunohistochemical markers p16, p53 and ki67.

BACKGROUND: Merkel cell polyomavirus (MCPyV), a human polyomavirus that is unequivocally linked to merkel cell carcinoma (MCC), has been found in association with keratinocytes carcinomas (KC), especially basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Nevertheless, there is scarce information about the possible involvement of MCPyV in the development of KC. OBJECTIVES: To assess the presence of MCPyV DNA and Large-T Antigen (LT-Ag) via Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) in cases of KC, and to correlate its presence with immunohistochemical markers p16, p53, and ki67, tumor type and subtype, sun-exposed location, and epidemiological data. METHODS: The prevalence of MCPyV DNA, LT-Ag, and immunohistochemical markers p16, p53, and ki67 was assessed by PCR and Immunohistochemistry (IHC) in 127 cases of KC, these results were correlated with tumor type and subtype, sun-exposed location, and epidemiological data. RESULTS: The MCPyV DNA was detected in 42.57% (43 of 101) cases by PCR, the LT-Ag was detected in 16.4% (20 of 122) of cases, p16 in 81.5% (97 of 119), p53 in 66.4% (83 of 125), ki67 in 89% (73 of 82). No correlation between MCPyV LT-Ag and DNA confronted with tumor type, subtype, location site, and immunohistochemical markers was found. A single correlation between the MCPyV LT-Ag and cSCC tumors and peri-tumoral lymphocyte cells was noted. STUDY LIMITATIONS: Further steps need to be taken to better evaluate the MCPyV influence and its possible role in KC carcinogenesis, as the evaluation of the virus genome state, the gene sequence that encodes LT-Ag in the KC tumor cells, and in situ hybridization for viral DNA or RNA in these cells. CONCLUSIONS: Despite the frequent detection of MCPyV in KC, the data available so far does not support the hypothesis of a causal relationship between them.

Reduction in disialyl-T antigen levels in mice deficient for both St6galnac3 and St6galnac4 results in blood filling of lymph nodes.

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galß1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.

Prevalence of human polyomaviruses in common and rare types of non-Merkel cell carcinoma skin cancer.

BACKGROUND: Little is known about the association of human polyomaviruses (HPyVs) other than Merkel cell polyomavirus (MCPyV) with nonmelanoma skin cancer. OBJECTIVES: To evaluate the presence of HPyV6, HPyV7, trichodysplasia spinulosa-associated polyomavirus (TSV), also called HPyV8, and the recently discovered HPyV9 in basal cell carcinoma (BCC), actinic keratosis (AK), squamous cell carcinoma in situ (SCCis), squamous cell carcinoma (SCC), keratoacanthoma (KA), microcystic adnexal carcinoma (MAC) and atypical fibroxanthoma (AFX). METHODS: Archival paraffin-embedded samples (n = 193: 41 BCC, 31 AK, 8 SCCis, 52 SCC, 42 KA, 5 MAC and 14 AFX) were analysed for the presence of the respective HPyV by polymerase chain reaction (PCR). HPyV DNA loads (HPyV DNA copies per ß-globin gene copy) were determined in all HPyV-positive samples by quantitative real-time PCR. Immunohistochemical analysis of MCPyV large T-antigen (LTA) expression was performed using the monoclonal antibody CM2B4. RESULTS: MCPyV DNA was found in 29% of BCC, 19% of AK, 25% of SCCis, 27% of SCC, 29% of KA, 0% of MAC and 29% of AFX. MCPyV DNA loads never exceeded 0·3 MCPyV DNA copies per ß-globin gene copy (median 0·004). In the immunohistochemical analysis of MCPyV LTA expression, all evaluated samples (32 MCPyV DNA-positive samples) were LTA negative. HPyV6 DNA was found in 7% of BCC, 3% of AK, 12% of SCCis, 4% of SCC, 5% of KA, and 0% of MAC and AFX. HPyV6 DNA loads never exceeded 0·7 HPyV6 DNA copies per ß-globin gene copy (median 0·015). None of the 193 samples was positive for HPyV7, TSV or HPyV9 DNA. CONCLUSIONS: Our findings argue against a pathogenic role for MCPyV, HPyV6, HPyV7, TSV and HPyV9 in the analysed types of non-Merkel cell carcinoma skin cancer.

No evidence for a causal role of Merkel cell polyomavirus in keratoacanthoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is a recently discovered virus that is monoclonally integrated into the genome of approximately 80% of all Merkel cell carcinomas (MCCs). While some evidence exists that MCPyV does not play a pathogenic role in other nonmelanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma (SCC), little is known about the presence of MCPyV in keratoacanthoma (KA). OBJECTIVES: To evaluate the prevalence, viral DNA-load, and large T(umor)-antigen expression of MCPyV in KA of immunocompetent patients and to compare the results with those found in SCC and MCC. METHODS: Paraffin-embedded tissue samples were analyzed for the presence of MCPyV-DNA by polymerase chain reaction (PCR). MCPyV-DNA load (MCPyV-DNA copies per beta-globin gene copy) was determined by using quantitative real-time PCR. Immunohistochemical analysis of the MCPyV large T-antigen was performed with the monoclonal antibody CM2B4. RESULTS: A total of 137 samples (42 KA, 52 SCC, and 43 MCC) were analyzed. MCPyV-DNA was found significantly more frequently in MCC (37/43, 86.0%) compared with KA (12/42, 28.6%) and SCC (14/52, 26.9%). Moreover, MCPyV-DNA loads were more than two orders of magnitude lower in KA and SCC compared with MCC (median/mean loads 0.005/0.015 [KA] vs 0.023/0.059 [SCC] vs 2.613/56.840 [MCC] MCPyV-DNA copies per beta-globin gene copy). All MCC analyzed (n = 3) expressed MCPyV large T-antigen, whereas 8 KA and 7 SCC were negative in immunohistochemistry. LIMITATIONS: The relatively small number of samples is a limitation. CONCLUSIONS: Our findings argue against a pathogenic role of MCPyV in KA and SCC.

Evidence of BK Polyomavirus Infection in Urothelial but not Renal Tumors from a Single Center Cohort of Kidney Transplant Recipients.

Emerging evidence indicates that reactivation of BK polyomavirus (BKPyV) in the kidney and urothelial tract of kidney transplant recipients (KTRs) may be associated with cancer in these sites. In this retrospective study of a single center cohort of KTRs (n = 1307), 10 clear cell renal cell carcinomas and 5 urinary bladder carcinomas were analyzed from 15 KTRs for the presence of BKPyV infection through immunohistochemistry and fluorescent in situ hybridization (FISH). Three of these patients had already exhibited biopsy-proven polyomavirus-associated nephropathies (PyVAN). Although the presence of BKPyV large-T antigen was evident in the urothelium from a kidney removed soon after PyVAN diagnosis, it was undetectable in all the formalin-fixed and paraffin-embedded (FFPE) blocks obtained from the 10 kidney tumors. By contrast, large-T antigen (LT) labeling of tumor cells was detected in two out of five bladder carcinomas. Lastly, the proportion of BKPyV DNA-FISH-positive bladder carcinoma nuclei was much lower than that of LT-positive cells. Taken together, our findings further strengthen the association between BKPyV reactivation and cancer development in KTRs, especially bladder carcinoma.

Expression of the 6C3 antigen on murine hematopoietic neoplasms. Association with expression of abl, ras, fes, src, erbB, and Cas NS-1 oncogenes but not with myc.

The monoclonal antibody 6C3 was used to test a wide variety of murine hematopoietic neoplasms for cell surface expression of a 160 kD glycoprotein (gp160(6C3)) previously shown to be expressed by neoplastic pre-B and some B lymphocytes transformed by Abelson murine leukemia virus (A-MuLV). This antigen was expressed on many pre-B and B cell lymphomas, but not on A-MuLV-transformed fibroblasts, T cell lymphomas, or myelomonocytic leukemias, gp160(6C3) was expressed by most early B-lineage spontaneous tumors, and early B tumors induced by replication-defective MuLV-containing oncogenes the products of which are associated with the cytoplasmic aspect of the plasma membrane, i.e., fes, abl, H-ras, bas, src, erbB, and Cas NS-1. By comparison, none of the early B lineage lymphomas induced by the "nuclear" oncogene avian v-myc MuLV, or arising in mice transgenic for a murine c-myc gene, or later B cell lineage stages bearing translocations of the c-myc locus expressed this antigen.

Transformed lymphocytes from Abelson-diseased mice express levels of a B lineage transformation-associated antigen elevated from that found on normal lymphocytes.

Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen-expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease.

Detection of human polyomavirus proteins, T-antigen and agnoprotein, in human tumor tissue arrays.

Expression of the human polyomavirus JCV genome in several experimental animals induces a variety of neural origin tumors. The viral proteins, T-antigen and Agnoprotein, contribute to the oncogenesis of JCV by associating with several tumor suppressor proteins and dysregulating signaling pathways, which results in uncontrolled cell proliferation. In addition, T-antigen and Agnoprotein have been associated with DNA damage and interfering with DNA repair mechanisms. In this study, we have utilized commercially available tissue arrays of human tumors of various origins and demonstrated the expression of both T-antigen and Agnoprotein in some, but not all, tumors of neural and non-neural origin. Most notably, more than 40% of human glioblastomas and greater than 30% of colon adenocarcinomas express viral proteins. The detection of viral transforming proteins, T-antigen and Agnoprotein in the absence of viral capsid proteins suggests a role for JCV in the development and/or progression of human tumors. These results invite further large-scale investigation on the role of polyomaviruses, particularly JCV in the pathogenesis of human cancer.

Prevalence of MCPyV in Merkel cell carcinoma and non-MCC tumors.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the likely causative agent of Merkel cell carcinoma (MCC). However, the prevalence of MCPyV in non-MCC population and its possible role in the pathogenesis of other skin cancers are not known yet. METHODS: A molecular pathology study was performed in 33 MCC samples and 33 age- and sex-matched samples of sun exposed non-MCC tumors [12 seborrheic keratoses (SK), 11 basal cell carcinomas (BCC) and 10 lentigo maligna melanomas (LMM)]. All tumors were analyzed for presence of MCPyV-DNA by polymerase chain reaction (PCR) and Southern-Blot hybridization of PCR products. RESULTS: MCPyV sequences were detected in 21 MCC samples (64%) and in 2 non-MCC tumors of sun exposed skin (6%; both SK-patients). Neither the tissue samples from BCC nor LMM proved positive for MCPyV sequences. CONCLUSION: We were able to confirm prior data on prevalence of MCPyV-DNA in MCC. Furthermore, a female predominance of MCPyV-positive MCC-patients was detected. There was no relevant association of MCPyV with SK, BCC and LMM. Speculative, prevalence of MCPyV in an age- and sex-matched non-MCC population could average up to 6%.

JC virus T-Antigen DNA in gastrointestinal mucosa of immunosuppressed patients: a prospective, controlled study.

BACKGROUND: JC virus (JCV), a polyoma virus, is the etiological agent of progressive multifocal leukoencephalopathy in immunosuppressed patients. JCV T-Ag has proven oncogenic potential and is expressed in colonic polyps and carcinomas. We proposed that the prevalence of JCV T-Ag DNA is higher in the normal gastrointestinal (GI) mucosa of immunosuppressed patients compared with their immunocompetent counterparts. AIMS: To look for JCV T-Ag DNA in the normal gastrointestinal mucosa of immunosuppressed patients compared with immunocompetent controls. METHODS: Macroscopically normal samples of upper and lower GI mucosa were obtained from 38 immunosuppressed patients. A control group included samples from 19 immunocompetent inflammatory bowel disease (IBD) and 29 non-IBD cases. DNA was extracted and polymerase chain reaction (PCR) was performed using primers specific for T-Ag. RESULTS: JCV T-Ag DNA was found in nine of the immunosuppressed patients (23.7%) and in three of the controls (6.3%; P = 0.02). Transplant recipients had a particularly high prevalence of JCV T-Ag DNA (35.3%). Patients with IBD receiving immunosuppressive drugs had a higher prevalence of JCV T-Ag DNA in comparison with IBD patients who did not receive immunosuppression (22.2% versus 10.5%, respectively), but this difference was not statistically significant (P = 0.574). CONCLUSION: JCV T-Ag DNA is more prevalent in the upper and lower GI mucosa of immunosuppressed patients, possibly indicating that the virus resides in these patients. This may account for the higher prevalence of GI carcinomas in immunosuppressed patients.

No implication of Simian virus 40 in pathogenesis of malignant pleural mesothelioma in Slovenia.

BACKGROUND AND AIM: Malignant mesothelioma is predominantly caused by asbestos exposure, although the association of Simian virus 40 in its pathogenesis is currently still under debate. Simian virus 40, a DNA rhesus monkey virus with oncogenic properties, accidentally contaminated early batches of polio vaccine in the 1960s. In the 1990s, viral sequences and proteins were discovered in several human tumors, which triggered research to find a link between Simian virus 40 and human cancers, especially malignant mesothelioma. The aim of our study was to establish an effective laboratory procedure for Simian virus 40 detection and to investigate the presence of Simian virus 40 DNA and small t antigen in mesothelioma samples from Slovenian patients. METHODS AND STUDY DESIGN: Paraffin-embedded malignant pleural mesothelioma specimens from 103 Slovenian patients were collected and used for total DNA isolation and real-time polymerase chain reaction for Simian virus 40 small t and large T DNA analysis. Special attention was devoted to primer design, good laboratory practice and polymerase chain reaction contamination prevention. Polymerase chain reaction products were sequenced and BLAST aligned. One 5 microm thick paraffin section from each patient's tissue block was stained with hematoxylin and eosin for histological typing and one for immunohistochemical detection of Simian virus 40 small t antigen using a monoclonal antibody against Simian virus 40 (Pab280). SV40-expressing Wi-38 cells were used as positive control in both PCR and immunohistochemistry. RESULTS: In real-time polymerase chain reaction analyses, only 4 samples gave products with primer pairs amplifying small t antigen and were inconsistent and poorly reproducible. BLAST alignment showed no homology with any deposited SV40 sequences. No immunopositive staining for SV40 small t antigen was found in any of the samples. CONCLUSIONS: We found no evidence of SV40 presence in tissue samples from 103 Slovenian patients with malignant pleural mesothelioma. Asbestos exposure remains the main risk factor for malignant pleural mesothelioma in Slovenia.

Soft tissue Rosai-Dorfman disease: 29 new lesions in 18 patients, with detection of polyomavirus antigen in 3 abdominal cases.

Soft tissue Rosai-Dorfman disease (STRDD) is rare, previously reported only as single cases and few series. Simian virus 40 (SV40), a polyomavirus, has been identified in lymphoid processes and has a controversial role in neoplasia etiology. Occasional cytoplasmic pink granular inclusions and nuclear changes led us to explore a viral etiology. Only unpublished STRDD from our files with adequate material, soft tissue location, and diagnostic confirmation were included. Immunohistochemistry and follow-up were obtained. Eighteen STRDD patients, 4 male and 14 female, had 29 lesions; 5 with 2 or more lesions. Ages ranged from 8 to 81 years (mean 42.6 years and median 42.5 years). Soft tissue Rosai-Dorfman disease locations include trunk or proximal extremity (n = 19), distal extremity (n = 5), "abdominal" (n = 3), face (n = 1), and unknown subcutaneous site (n = 1). Sizes ranged from 0.5 to 13.7 cm (median, 2.4 cm). Previous disease included lymphoma, buttocks injection site, diabetes and hypothyroidism, and radiation for chronic dermopathy. No patients had a preceding or concurrent known viral infection; none had lymphadenopathy at present. None were known to be immunocompromised. Soft tissue Rosai-Dorfman disease was rapidly progressing. Initial pathologic diagnosis ranged from Rosai-Dorfman disease or inflammatory pseudotumor to inflammatory malignant fibrous histiocytoma. Grossly STRDDs were multilobulated, tan-yellow, and firm; morphologically, circumscribed, and subcutaneous-based. All had sheets of polygonal histiocytes with abundant pale eosinophilic cytoplasm, emperipolesis, plasma cells, and lymphocytes scattered and within clusters. Focal spindle cell change and mild pleomorphism were each observed in 3 patients; 2 had focal necrosis, none with mitoses. Small granular pink cytoplasmic inclusions and nuclear viral-like changes were observed. By immunohistochemistry, all STRDDs were positive for S100 protein, negative for CD1a, Epstein-Barr virus, and latent membrane protein, yet 3 (all abdominal, 1 multicentric) of the 9 studied were focally positive for cytoplasmic and nuclear SV40 polyomavirus. All were treated by local excision. Follow-up on 14 patients older than 8 to 16 years revealed recurrence in 3 patients with persistent multiple lesions, one with abdominal location. There were no metastases or death from disease. Soft tissue Rosai-Dorfman disease is a rapidly evolving, mostly solitary and nonrecurrent trunk and proximal extremity subcutaneous lesion in middle-aged females. More than one third can have persistent multicentric disease. It is important to recognize STRDD, to separate it from malignancy. Epstein-Barr virus/latent membrane protein was negative but polyomavirus was positive in 3 patients with abdominal STRDD, one with multicentric persistent disease. The relationship of polyomavirus to the evolution of abdominal STRDD should be further explored.

Comparison of Immunohistochemical Staining for Large T Antigen and Capsid Protein VP1 in BK Polyomavirus-Associated Nephropathy.

AIM: Most transplant centres use SV40 large T antigen (TAg) staining for the diagnosis and assessment of BK polyomavirus-associated nephropathy (BKPyVAN). This study was performed to evaluate the significance of capsid protein VP1 expression in BKPyVAN. METHODS: We performed immunohistochemical staining using anti-SV40 TAg and anti-BKPyV VP1 antibodies in 16 index biopsies and 12 re-biopsies of BKPyVAN and compared the patterns of positivity and the percentage of positive tubules by counting whole specimens. We investigated the correlation between serum creatinine increase from baseline and the percentage of positive tubules for both markers in 16 index biopsies. RESULTS: In VP1 staining, positive findings were observed not only in the nuclei of tubular epithelial cells but also in the cytoplasm, cells shedding into the lumen, intra-tubular casts, and in the interstitium. Two of 28 biopsies (7.1%) showed TAg-positive and VP1-negative results, in which TAg-positive cells were detected only in a single tubule. The median (interquartile range) percentage of positive tubules was 2.8% (0.7-9.8%) for TAg and 1.4% (0.5-3.9%) for VP1 staining (p = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (r = 0.49, p = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. CONCLUSIONS: VP1 expression showed various patterns, but was detected in half as many tubules as TAg staining, which might lead to false negatives in the samples with minimal viral replication. However, increased VP1-positive tubules indicate advanced tubular damage and possible association with graft dysfunction.

A cytoplasmically anchored nuclear protein interferes specifically with the import of nuclear proteins but not U1 snRNA.

A cytoplasmically anchored mutant SV40 T antigen, FS T antigen, was shown previously to interfere specifically with the nuclear import of a heterologous nuclear protein, adenovirus 5 fiber protein, in cultured monkey cells (Schneider, J., C. Schindewolf, K. van Zee, and E. Fanning. 1988. Cell. 54:117-125; van Zee, K., F. Appel, and E. Fanning. 1991. Mol. Cell. Biol. 11:5137-5146). In this report, we demonstrate that FS T antigen also interferes with the nuclear import of adenovirus E1A and a peptide-albumin conjugate bearing multiple copies of the T antigen nuclear localization signal, but not with the import of U1 snRNA. A kinetic analysis indicates that nuclear import of the albumin-peptide conjugate is inhibited only when high intracellular concentrations of FS T antigen are reached. After microinjection into the cytoplasm of cultured cells, purified FS T antigen protein does not accumulate at the nuclear periphery, but rather is distributed in a punctate pattern throughout the cytoplasm. These data support a model in which cytoplasmic anchoring of FS T antigen enables the mutant protein to sequester and titrate out a cellular factor which is required for nuclear protein but not U1 snRNA import.

Oncogenesis of the lens in transgenic mice.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.

The role of individual cysteine residues in the structure and function of the v-sis gene product.

The v-sis oncogene encodes a platelet-derived growth factor (PDGF)-related product whose transforming activity is mediated by its functional interaction with the PDGF receptor. PDGF, as well as processed forms of the v-sis gene product, is a disulfide-linked dimer with eight conserved cysteine residues in the minimum region necessary for biologic activity. Site-directed mutagenesis of the v-sis gene revealed that each conserved cysteine residue was required directly or indirectly for disulfide-linked dimer formation. However, substitution of serine for cysteine codons at any of four positions had no detrimental effect on transforming activity of the encoded v-sis protein. These results establish that interchain disulfide bonds are not essential in order for this protein to act as a functional ligand for the PDGF receptor. The remaining four substitutions of serine for cysteine each inactivated transforming function of the molecule. In each case this was associated with loss of a conformation shown to involve intramolecular disulfide bonds. These studies provide insight into the role of individual cysteine residues in determining the structure of the sis/PDGF molecule critical for biological activity.

JC virus infection in colorectal neoplasia that develops after liver transplantation.

PURPOSE: Liver transplant recepients (LTRs) have an increased risk of colorectal neoplasia. The mechanism responsible for this is unknown. JCV encodes for TAg and has been implicated in colorectal carcinogenesis. We hypothesized that the use of immunosuppression in LTRs facilitates activation of JCV and is responsible for the increased risk of neoplasia. EXPERIMENTAL DESIGN: JCV TAg DNA and protein expression were determined in normal colonic epithelium (n = 15) and adenomatous polyps (n = 26) from LTRs and compared with tissue samples from control patients (normal colon, n = 21; adenomas, n = 40). Apoptosis and proliferation were determined by M30 and Ki-67 immunoreactivity, respectively. RESULTS: JCV TAg DNA was found in 10 of 15 (67%) of normal colonic mucosa from LTRs compared with 5 of 21 (24%) of control normal mucosa (P = 0.025). JCV TAg DNA was detected in 16 of 26 (62%) of the adenomas from LTRs and in 20 of 40 (50%) of control adenomas. JCV TAg protein was expressed in 13 of 26 (50%) adenomas from LTRs versus 2 of 40 (5%) of adenomas from controls (P < 0.001). In adenomas from LTRs, the mean proliferative activity was higher compared with controls (60.3 +/- 3.2% versus 42.7 +/- 2.8%, P < 0.001), whereas mean apoptotic indices were lower in LTRs (0.29 +/- 0.08% versus 0.39 +/- 0.06%, P = 0.05). CONCLUSIONS: The presence of JCV in the colorectal mucosa and adenomas from LTRs, in concert with the use of immunosuppressive agents, suggests that JCV may undergo reactivation, and the subsequent TAg protein expression might explain the increased risk of colorectal neoplasia in LTRs.

Polyoma virus infection and urothelial carcinoma of the bladder following renal transplantation.

Renal transplant recipients are at increased risk of bladder carcinoma. The aetiology is unknown but a polyoma virus (PV), BK virus (BKV), may play a role; urinary reactivation of this virus is common post-renal transplantation and PV large T-antigen (T-Ag) has transforming activity. In this study, we investigate the potential role of BKV in post-transplant urothelial carcinoma by immunostaining tumour tissue for PV T-Ag. There was no positivity for PV T-Ag in urothelial carcinomas from 20 non-transplant patients. Since 1990, 10 transplant recipients in our unit have developed urothelial carcinoma, and tumour tissue was available in eight recipients. Two patients were transplanted since the first case of PV nephropathy (PVN) was diagnosed in our unit in 2000 and both showed PV reactivation post-transplantation. In one of these patients, there was strong nuclear staining for PV T-Ag in tumour cells, with no staining of non-neoplastic urothelium. We conclude that PV infection is not associated with urothelial carcinoma in non-transplant patients, and is uncommon in transplant-associated tumours. Its presence in all tumour cells in one patient transplanted in the PVN era might suggest a possible role in tumorigenesis in that case.