Expression of Thymidine Phosphorylase in Lymph Nodes Involved with Mycosis Fungoides and Sézary Syndrome

Thymidine phosphorylase may be overexpressed in both neoplastic cells and tumor stromal cells in a variety of malignancies. Our study explores thymidine phosphorylase expression in lymph nodes (LNs) from patients with mycosis fungoides (MF) or Sézary syndrome (SS). In MF/SS, the LNs may have a pathologic diagnosis of either dermatopathic lymphadenopathy (LN-DL) or involvement by MF/SS (LN-MF). We performed immunohistochemical staining on MF/SS lymph nodes using antibodies to thymidine phosphorylase, CD68, CD21, CD3, and CD4. In both LN-DL and benign nodes, thymidine phosphorylase staining was noted only in macrophages, dendritic cells, and endothelial cells. In LN-MF, thymidine phosphorylase expression was also noted in subsets of intermediate to large neoplastic T cells. Concurrent CD68, CD21, CD3, and CD4 staining supported the above observations. Similar results were noted in the skin and in LN-MF with large cell transformation. Other T-cell lymphomas were also examined (total 7 cases); only enteropathy-type T-cell lymphoma (1 case) showed TP positivity in neoplastic T lymphocytes. We demonstrated that thymidine phosphorylase staining is present in neoplastic T cells in mycosis fungoides. The exact mechanism needs further investigation

Action of Inhibitors on Accumulation of Processed Hepatitis Delta Virus RNAs

Hepatitis delta virus (HDV) replication involves processing and accumulation of three RNA species: the genome, its exact complement (the antigenome), and a polyadenylated mRNA that acts as a template for the small delta antigen (δAg), the only protein of HDV and essential for genome replication. In a recently reported experimental system, addition of tetracycline induced synthesis of a DNA-directed source of δAg, producing within 24 h a significant increase in accumulation of newly transcribed and processed HDV RNAs. This induction was used here to study the action of various inhibitors on accumulation. For example, potent and HDV-specific inhibition, in the absence of detected host toxicity, could be obtained with ribavirin, mycophenolic acid, and viramidine. An interpretation is that these inhibitors reduced the available GTP pool, leading to a specific inhibition of the synthesis and accumulation of HDV RNA-directed RNA species. In contrast, no inhibition was observed with l-FMAU (2′-fluoro-5-methyl-β-l-arabinofuranosyl-uridine), alpha interferon, or pegylated alpha interferon. After modifications to the experimental system, it was also possible to examine the effects of three known host RNA polymerase inhibitors on HDV genome replication: amanitin, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), and actinomycin. Of most interest, amanitin at low doses blocked accumulation of HDV RNA-directed mRNA but had less effect on HDV genomic and antigenomic RNAs. Additional experiments indicated that this apparent resistance to amanitin inhibition of genomic and antigenomic RNA relative to mRNA may not reflect a difference in the transcribing polymerase but rather relative differences in the processing and stabilization of nascent RNA transcripts

Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II▿

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories

Development of a Novel System To Study Hepatitis Delta Virus Genome Replication

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (δAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of δAg species that are nonfunctional or even inhibitory. A replication system was devised where all δAg was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small δAg, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express δAg. Thus, replication of this RNA was under control of the TET-inducible δAg. In the absence of TET, there was sufficient δAg to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both δAg and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G(1)/G(0) cells relative to both S and G(2)/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system

Thymidine phosphorylase expression in B-cell lymphomas and its significance: a new prognostic marker?

OBJECTIVE: To explore thymidine phosphorylase (TP) expression in B-cell lymphomas (BCLs). TP is expressed by tumor and stromal cells in a variety of cancers. STUDY DESIGN: Paraffin-embedded tissues from follicular lymphomas, diffuse large BCLs (DLBCLs), and benign lymph nodes were studied using immunohistochemical staining with antibodies for TP and CD68. Prognostic markers were used to stain DLBCLs. We correlated TP expression in DLBCL indirectly with prognostic immunomarkers and directly with survival data. RESULTS: TP expression in BCLs was noted in a subset of malignant B cells. TP expression in higher-grade lymphoma was identified in 66% of cases and 11% of lower-grade lymphomas. Macrophages/stromal cells demonstrated an intense cytoplasmic and/or nuclear staining pattern in both lymphoma and benign lymph nodes, confirmed by CD68 coexpression. Increased macrophage/ stromal cells in higher-grade lymphomas are associated with enhanced TP expression in neoplastic B cells (observation only). Sixty-eight percent of TP-positive DLBCLs were of nongerminal center origin, indicating poorer prognosis. CONCLUSION: TP is more likely expressed by malignant B cells in higher-grade lymphomas, and expression of TP possibly results from changes intrinsic to the tumor cells or interactions between microenvironment and tumor. TP positivity in DLBCL correlates with nongerminal center origin and worse outcome