Lineage pathway of human brain progenitor cells identified by JC virus susceptibility

Multipotential human central nervous system progenitor cells, isolated from human fetal brain tissue by selective growth conditions, were cultured as undifferentiated, attached cell layers. Selective differentiation yielded highly purified populations of neurons or astrocytes. This report describes the novel use of this cell culture model to study cell type-specific recognition of a human neurotropic virus, JC virus. Infection by either JC virions or a plasmid encoding the JC genome demonstrated susceptibility in astrocytes and, to a lesser degree, progenitor cells, whereas neurons remained nonpermissive. JC virus susceptibility correlated with significantly higher expression of the NFI-X transcription factor in astrocytes than in neurons. Furthermore, transfection of an NFI-X expression vector into progenitor-derived neuronal cells before infection resulted in viral protein production. These results indicate that susceptibility to JC virus infection occurs at the molecular level and also suggest that differential recognition of the viral promoter sequences can predict lineage pathways of multipotential progenitor cells in the human central nervous system. Neurol 2003;53:636 -646 The differentiation of central nervous system (CNS) stem and progenitor cells into neuronal and glial lineages is accompanied by the expression of specific intracellular molecules involved in the transcription of cell type-specific genes. The selective differentiation of human CNS progenitor cells into neural cell types provides a unique model to study the molecular regulation of cellular phenotypes as well as neurotropic viruses that target specific subpopulations of CNS cells. For example, the human polyomavirus, JC virus (JCV), demonstrates a restricted cellular host range and tropism in the CNS, targeting glial but not neuronal cells. Ann 1,2 Lytic infection of oligodendrocytes results in the fatal demyelinating disease, progressive multifocal leukoencephalopathy. 1,2 JCV infects astrocytes both in vivo and in vitro but does not infect neuronal cells, JCV is unique among most viruses in that viral binding and entry do not predict susceptibility to infection. 7 Therefore, it is hypothesized that the selective tropism of JCV is governed by molecular determinants, namely, nuclear transcription factors located within susceptible cells. The promoter-enhancer region of JCV contains multiple sites for the nuclear factor-1 (NFI) family of transcription factors, 8 -10 which includes four members, NFI-A, NFI-B, NFI-C, and NFI-X 11,12 (also known as NFI-D). NFI has been implicated in the transcriptional regulation of several CNS-specific cellular genes 12-17 and viral replication, 18 -21 including that of JCV. The human CNS multipotential progenitor cells, described in this study, were used as a unique in vitro model to study the molecular regulation of JCV infection and to examine the potential role of NFI transcription factors in initiating viral multiplication in specific subpopulations of CNS cell types. The data presented in this article are the first to our knowledge demonstrating JCV infection of a population of human CNS progenitor cells. These experiments indicate that susceptibility to infection depends not on viral binding and entry, but on intracellular factors. Notably, overexpression of one of the NFI class members, NFI-X, in the neuronal cells initiated JCV susceptibility. These data substantiate the importance of NFI-X recognition in the transcriptional regulation of JCV susceptibility From th

Thrombopoietin Receptor Levels in Tumor Cell Lines and Primary Tumors

Thrombopoietin (TPO) receptor agonists represent a new approach for the treatment of thrombocytopenia, which may develop as a consequence of immune thrombocytopenia, chemotherapy treatment, chronic hepatitis C infection, or myelodysplastic syndromes. There are concerns that use of certain growth factors can hasten disease progression in some types of hematologic malignancies and solid tumors. In this study, expression of MPL (TPO-R) mRNA was examined in tumor cell lines, patient tumor samples (renal cell carcinoma, prostatic carcinoma, soft tissue and bony/cartilage sarcoma, colon cancer, and lymphoma), and normal tissues using microarray analysis and qRT-PCR. MPL mRNA is expressed at very low or undetectable levels compared with erythropoietin receptor (EPOR), human epidermal growth factor (ERBB2; HER2), and insulin-like growth factor-1 receptor (IGF1R) in these patient samples. These data suggest TPO-R agonists will likely preferentially stimulate proliferation and differentiation of cells of megakaryocytic lineage, potentially demonstrating their utility for correcting thrombocytopenia in clinical settings

Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors

BACKGROUND: Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. METHODS: Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. RESULTS: MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. CONCLUSIONS: Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors

Cellular and biochemical changes occurring during apoptosis of PC12 cells

The goal of this thesis project was to address several features of apoptosis that had not been investigated previously. These included: (1) defining the sequence of morphological changes accompanying apoptosis so that morphological markers can be used in future studies as reference points to place cellular and biochemical changes within a temporal sequence of events, (2) determining if all cells in a population undergo similar morphological changes during apoptosis, (3) determining at what point following an apoptotic stimulus cells commit to die, (4) determining if commitment to die can be defined relative to characteristic morphological changes, and (5) determining a role for intracellular Ca\sp{2+} in modulating the execution phase of apoptosis in PC12 cells. Results from time lapse video microscopy experiments indicate that all cells in a population undergo similar and clearly defined morphological changes following an apoptotic stimulus even if they enter the execution phase of apoptosis more than 48 hrs apart. Since the sequence of morphological changes cells go through are stereotyped and very consistent from cell to cell, they can be used as reference points to place other cellular events. The point cells commit to die occurs 2-3 hrs prior to entering the execution phase of apoptosis. Video microscopy data indicate that apoptosis is an inherently asynchronous process even when daughter cells are synchronized following mitosis. Measurement of intracellular free Ca\sp{2+} in individual apoptotic cells showed a sustained elevation of Ca\sp{2+} occurring 1-2 hrs before cells die. This increased intracellular free Ca\sp{2+} correlates temporally with the onset of the execution phase. To determine if the rise in Ca\sp{2+} affected processes in the execution phase, cells were transfected with the Ca\sp{2+} binding protein, calbindin, or loaded with the Ca\sp{2+} chelator, BAPTA. The execution phase appears to be a highly conserved feature of apoptosis; therefore, it is surprising that its length is considerably shorter in cells expressing calbindin or in cells loaded with BAPTA. Results obtained with calbindin expressing cells and BAPTA loaded cells are consistent with the length of the execution phase of apoptosis being regulated by intracellular free Ca$\sp{2+}.