Association of SRC-related kinase Lyn with the interleukin-2 receptor and its role in maintaining constitutive phosphorylation of JAK/STAT in human T-cell leukemia virus type 1-transformed T cells.

Human T-cell leukemia virus type 1 (HTLV-1) infection and transformation are associated with an incremental switch in the expression of the Src-related protein tyrosine kinases Lck and Lyn. We examined the physical and functional interactions of Lyn with receptors and signal transduction proteins in HTLV-1-infected T cells. Lyn coimmunoprecipitates with the interleukin-2 beta receptor (IL-2Rß) and JAK3 proteins; however, the association of Lyn with the IL-2Rß and Lyn kinase activity was independent of IL-2 stimulation. Phosphorylation of Janus kinase 3 (JAK3) and signal transducers and activator of transcription 5 (STAT5) proteins was reduced by treatment of cells with the Src kinase inhibitor PP2 or by ectopic expression of a dominant negative Lyn kinase protein.

David D. Derse, 1949-2009.

David D. Derse, Ph.D., Head of the Retrovirus Gene Expression Section in the HIV Drug Resistance Program at the National Cancer Institute-Frederick (NCI-Frederick), passed away on October 9, 2009, a scant six weeks after being diagnosed with liver cancer. It was with great sadness that family, friends, and colleagues gathered together for his memorial service on Saturday, October 17, 2009, at the Middletown United Methodist Church in Maryland. As a NCI scientist since 1986, Dave studied the molecular mechanisms of infection and replication of a number of different types of retroviruses. Dave became an internationally known expert on human T cell lymphotrophic viruses type 1 and 2 (HTLV-1 and HTLV-2) and served on the editorial boards of Virology and Retrovirology. His most recent studies focused on the mechanisms of HTLV-1 virion morphogenesis, transmission, and replication.

A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis.

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis. To develop a patient-derived orthotopic xenograft model to mimic clinical urothelial cell carcinoma progression to metastatic disease, the urothelial cell carcinoma cell line UM-UC-3 and two urothelial cell carcinoma patient specimens were doubly tagged with Luciferase/RFP and were intra-vesically (IB) instilled into NOD/SCID mice with or without lymph node stromal cells (HK cells). Mice were monitored weekly with bioluminescence imaging to assess tumor growth and metastasis. Primary tumors and organs were harvested for bioluminescence imaging, weight, and formalin-fixed for hematoxylin and eosin and immunohistochemistry staining. In this patient-derived orthotopic xenograft model, xenograft tumors showed better implantation rates than currently reported using other models. Xenograft tumors histologically resembled pre-implanted primary specimens from patients, presenting muscle-invasive growth patterns. In the presence of HK cells, tumor formation, tumor angiogenesis, and distant organ metastasis were significantly enhanced in both UM-UC-3 cells and patient-derived specimens. Thus, we established a unique, reproducible patient-derived orthotopic xenograft model using human high-grade urothelial cell carcinoma cells and lymph node stromal cells. It allows for investigating the mechanism involved in tumor formation and metastasis, and therefore it is useful for future testing the optimal sequence of conventional drugs or the efficacy of novel therapeutic drugs.

Negligible Oval Cell Proliferation Following Ischemia-Reperfusion Injury With and Without Partial Hepatectomy.

BACKGROUND: Hepatic oval cells proliferate to replace hepatocytes and restore liver function when hepatocyte proliferation is compromised or inadequate. Exposure to chemical carcinogens, severe liver steatosis, and partial hepatectomy has been used in animal models to demonstrate the role of oval cells in liver regeneration. Ischemia-reperfusion injury (IRI) causes hepatocellular damage and death in the absence of confounding chemical toxicity; however, oval cell induction by IRI has not been demonstrated in vivo. We examine oval cell induction following partial IRI. METHODS: Wistar rats were subjected to 2 IRI protocols: 70% warm liver ischemia for 30 minutes followed by reperfusion or 70% warm liver ischemia for 30 minutes with partial hepatectomy of the nonischemic lobes followed by reperfusion. Liver injury was monitored by serum alanine aminotransferase (ALT) at 1 day and 7 days of reperfusion. Oval cell proliferation was monitored by indirect immunofluorescence staining using the surface markers BD.2 and Thy-1. Cellular proliferation was quantified by 5-ethynyl-2'-deoxyuridine (EdU) incorporation in vivo. RESULTS: Serum ALT elevation was only observed at the 1-day time point in the IRI with partial hepatectomy model. Oval cell marker expression was restricted to the biliary structures in both the ischemic and the nonischemic control lobes. Oval cell induction, measured by changes in the frequency of BD.2 and Thy-1 expression and EdU incorporation, was not significantly altered by IRI. CONCLUSION: In both mild and moderate IRI models, we did not find evidence of oval cell induction or proliferation. EdU staining was restricted to hepatocytes, suggesting that liver regeneration following IRI is mediated by hepatocyte proliferation.

V(D)J recombination of chromosomally integrated, wild-type deletional and inversional substrates occur at similar frequencies with no preference for orientation.

Efficient and correct recombination of V(D)J substrates results in the generation of antibodies. The RSS substrates are oriented in two directions with respect to each other: deletional and inversional. Deletional recombination results in the formation of the coding joint and excision of the intervening sequences. Inversional recombination retains all the genomic sequences and forms both a coding joint and a signal joint. A bias for deletional recombination has been characterized with specific loci in vivo and recapitulated in experiments using extrachromosomal substrates. We constructed retroviral substrates of RSS in the deletional and inversional orientation. We introduced the substrates into wild-type and scid pre-B cells and measured the frequency of functional recombination in addition to open/shut recombination. We also mutated the RSSs to determine whether mutated sequences influenced orientation bias. We show that pre-B cells recombine the wild-type substrates at a 1.6 ratio of deletion:inversion. Nonamer mutated substrates recombined with a deletional bias whereas heptamer mutated substrates recombined with an inversional bias. A spacer length mutation and drastic mutations in the RSS abolish all recombination. These results suggest that there is no orientation bias with wild-type RSSs but that orientation bias occurs when RSSs are mutated.

The human T-cell leukemia virus type 1 (HTLV-1): new insights into the clinical aspects and molecular pathogenesis of adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM).

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be identified in the early 1980s. The isolation and identification of a related virus, HTLV-2, and the distantly related human immunodeficiency virus (HIV) immediately followed. Of the three retroviruses, two are associated definitively with specific diseases, HIV, with acquired immune deficiency syndrome (AIDS) and HTLV-1, with adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). While an estimated 10-20 million people worldwide are infected with HTLV-I, infection is endemic in the Caribbean, parts of Africa, southwestern Japan, and Italy. Approximately 4% of HTLV-I infected individuals develop ATLL, a disease with a poor prognosis. The clinical manifestations of infection and the current biology of HTLV viruses with emphasis on HTLV-1 are discussed in detail. The implications for improvements in diagnosis, treatment, intervention, and vaccination are included, as well as a discussion of the emergence of HTLV-1 and -2 as copathogens among HIV-1-infected individuals.

Tumor Necrosis Factor-α: Life and Death of Hepatocytes During Liver Ischemia/Reperfusion Injury.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine involved in a variety of disease pathologies, including ischemia/reperfusion (I/R) injuries in transplantation. The interaction of TNF-α with its cognate receptor TNF receptor I (TNFRI) results in the activation of signal transduction pathways that regulate either cell survival or cell death. Hepatocytes express TNFRI and respond to TNF-α released by resident Kupffer cells as well as leukocytes that migrate to the liver during I/R injury. Upon binding TNF-α, the hepatocyte proliferates or undergoes apoptosis or necroptosis. The decision by the cell to commit to one path or the other is not understood. The damaged tissue exhibits cell death and hemorrhaging from the influx of immune mediators. TNF-α inhibitors ameliorate the injury in animal models, suggesting that lowering (but not eliminating) TNF-α levels shifts the balance of TNF-α toward its beneficial functions. METHODS: We review TNF-α signal transduction pathways and the role of TNF-α in liver I/R injury. CONCLUSIONS: Because TNF-α plays an important role in hepatocyte proliferation, complete inhibition of TNF-α is not desirable in treating liver I/R injury. The strategy for developing pharmacological therapies may be the identification of specific intermediates in the TNF-α/TNFR1 signal transduction pathway and directed targeting of proapoptotic and pronecroptotic events.