We have previously reported (O'Garra, A. et al., Int. Immunol. 1990, 2:821) that murine B lymphomas and purified normal peritoneal B cells produce interleukin (IL) 10. We now show that this production of IL 10 B cells correlates with the presence of Ly-1 (B-1) B cells, in both normal and diseased mice. Using a semi-quantitative modification of the polymerase chain reaction, we show that IL 10 expression is detectable in peritoneal B cells but only becomes apparent in splenic B cells of aged mice of which a high proportion are Ly-1+. Furthermore, the expression of IL 10 is constitutive in splenic B cells from mice carrying the Ly-1+ BCL1 lymphoma. Since IL 10 is a potent regulator of in vitro immune function, its production by Ly-1 lineage B (B-1) cells raises the possibility that this subset of B cells may regulate their own development and/or the function of other immunocompetent cells.

SummaryHighly purified, small dense splenic B cells from unstimulated mice showed increased expression ofclass II major histocompatibility complex (MHC) antigens and enhanced viability when cultured with affinity-purified recombinant interleukin 10 (HIA0), compared with B cells cultured in medium alone. These responses were blocked by a monoclonal antibody (mAb) specific for 11,10, but not by an isotype-matched control antibody. IL10 did not upregulate the expression of Fce receptors (CD23) or class I MHC antigens on small dense B cells or induce their replication as monitored by [3H]thymidine incorporation. While these B cell-stimulatory properties of IWO are also mediated by ID4, the two cytokines appear to act independently in these assays ; anti-IL10 antibodies blocked 1140 but not I14-mediated B cell viability enhancement, and vice versa. Similarly, since IIr4 upregulates CD23 on small dense B cells, the inability of IIr10 to do so argues against its acting via endogenously generated 114. Finally, IIA0 did not upregulate class II MHC antigens on B cells from X chromosome-linked immunodeficiency (XID) mice, while the same cells showed normal upregulation of class II antigens in response to 11.4. This report also extends our understanding of the relationship between IIA0 and the highly homologous Epstein-Barr virus (EBV)-encoded Barn HI fragment C rightward reading frame no. 1 (BCRFI) protein. It has previously been shown that BCRFI protein exhibits the cytokine synthesis inhibitory activity of IL10. This report indicates that BCRFI protein also enhances in vitro B cell viability, but does not upregulate class II MHC antigens on B cells. One explanation for these data is that 111,10 contains at least two functional epitopes, only one ofwhich has been conserved by EBV Blymphocytescontribute to the immune response by their production of specific antibodies in response to antigenic stimuli. This process is regulated by a subset of soluble glycoproteins collectively termed cytokines (1-3). The precise number of cytokines involved and their modes of action require further clarification. 11,10 (originally designated cytokine synthesis inhibitory factor [CSIF]l; 4) is a cytokine produced by activated type 2 T helper (Th2) cells (4, 5) and B cells (6, 7), which has the property ofsuppressing cytokine production by type 1 T helper (Thl) cells (4). Isolation of the cDNA for mouse RAO (8) revealed a striking homology between ' Abbreviations used in this paper: BCRFI, BamHI fragment C rightward reading frame no. 1 ; CSIF, cytokine synthesis inhibitory factor; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SA, streptavidin; Thl/Th2, type 1/type 2 T helper cells; XID, X chromosome-linked immunodeficiency. this cytokine and a previously uncharacterized open reading frame, designated BamHI fragment C rightward reading frame no. 1 (BCRFI), in the EBV genome. Subsequent expression and testing of the protein encoded by BCRFI revealed that this molecule also mediated suppression of Thl cytokine production (...

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy. Similar to other malignant human tumor types, 4 over 80% of human HCC biopsies show activation of telomerase. 5 In contrast, most somatic human tissues, including normal liver, 6 show no or very low levels of telomerase activity. The main function of telomerase is the de novo synthesis of telomeres, which cap the chromosome ends of eukaryotic cells and protect chromosome ends from fusion and DNA damage recognition. 7 Because of the "end replication problem" of DNA polymerase, telomeres shorten during each cell division by 50 to 100 bp. 8 Telomere shortening to a critical length and/or the uncapping of the telomere limit the growth of primary human cells to a finite number of cell divisions, leading to either replicative senescence or crisis. 9-11 A critically short telomere

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after <4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice, concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy.