Exploring the native human antibody repertoire to create antiviral therapeutics.

Native human antibodies are defined as those that arise naturally as the result of the functioning of an intact human immune system. The utility of native antibodies for the treatment of human viral diseases has been established through experience with hyperimmune human globulins. Native antibodies, as a class, differ in some respects from those obtained by recombinant library methods (phage or transgenic mouse) and possess distinct properties that may make them ideal therapeutics for human viral diseases. Methods for cloning native human antibodies have been beset by technical problems, yet many antibodies specific for viral antigens have been cloned. In the present review, we discuss native human antibodies and ongoing improvements in cloning methods that should facilitate the creation of novel, potent antiviral therapeutics obtained from the native human antibody repertoire.

hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase.

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. We propose that hSir2 is involved in the regulation of p53 function via deacetylation.

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Methylation of the human telomerase gene CpG island.

The acquisition of expression of hTERT, the catalytic subunit of the telomerase enzyme, seems to be an essential step in the development of a majority of human tumors. However, little is known about the mechanisms preventing telomerase gene expression in normal and transformed cells that do not express hTERT. Using a methylation-specific PCR-based assay, we have found that the CpG island associated with the hTERT gene is unmethylated in telomerase-negative primary tissues and nonimmortalized cultured cells, indicating that mechanisms independent of DNA methylation are sufficient to prevent hTERT expression. The hTERT CpG island is methylated in many telomerase-negative and telomerase-positive cultured cells and tumors, but the extent of methylation did not correlate with expression of hTERT. Demethylation of DNA with 5-azacytidine in two cell lines induced expression of hTERT, suggesting that DNA methylation can contribute to hTERT repression in some cells. Together, these data show that the hTERT CpG island can undergo cytosine methylation in cultured cells and tumors and that DNA methylation may contribute to the regulation of the hTERT gene, but that CpG island methylation is not responsible for repressing hTERT expression in most telomerase-negative cells.

N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma.

Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases in the in vivo growth rate and metastatic ability of these cells. N-myc-mediated down-modulation of MHC class I antigen expression could be reversed by treatment with interferon without affecting the steady state level of N-myc mRNA. No effect on MHC class I antigen expression was found when the N-myc gene was expressed in rat fibroblasts, indicating that some of the effects caused by N-myc gene amplification are cell-type-specific.