Most cancer therapies involve a component of treatment which inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radio-sensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules which selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits which potently inhibit DSB repair, and we have validated their functional activity in comprehensive panel of orthogonal secondary assays. A selection of these inhibitors were found to radiosensitize cancer cell lines in vitro, which suggests they may be useful as novel chemo- and radio-sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker, mibefradil dihydrochloride, which demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a Phase I clinical trial testing mibefradil as glioma radiosensitizer.

Self-renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short- and long-term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF-like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non-adherent co-cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway.

Human Jurkat T-cell clones containing stably integrated HIV-1 LTR or HTLV-1 LTR/lacZ vectors were studied to compare the responses of integrated LTRs to T-cell activation. Responses were compared also with those obtained in parallel with Jurkat cells stably expressing lacZ under the control of the cellular enhancer element NF-AT of the IL-2 promoter. Activation induced via the cell surface TCR/CD3 complex or the CD28 receptor elicited responses from the LTR of HIV-1; however, HTLV-1 LTR-directed expression was not observed following triggering of these cell surface pathways. Mitogenic activation by elevation of intracellular calcium (Ca2+) levels along with protein kinase C (PKC) signals was required for optimal expression of the HIV-1 LTR and the NF-AT element; however, increased intracellular Ca2+ was inhibitory to PKC-mediated expression from the HTLV-1 LTR. Time course experiments revealed a sustained PKC-mediated response by the HTLV-1 LTR, which was detectable in the absence of Ca2+ as early as 6 hr following stimulation. In contrast to the HTLV-1 LTR, in time course experiments the HIV-1 LTR responded to stimulation by mitogenic activation of PKC in the absence and presence of Ca2+ and by antiCD3 with lacZ expression beginning as early as 3 hr poststimulation. These results suggest that the HTLV-1 LTR appears to be refractory to several cellular pathways which are upregulatory to the HIV-1 LTR.

A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples. 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay. D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity. With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells.

Femora and tibiae of rats carrying leukemia from a L a dmarked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections IntroductionIn acute myelocytic leukemia (AML). infiltration of the bone marrow with leukemic cells is accompanied by severe suppression of normal hematopoiesis, eventually leading to life-threatening bleeding problems, recurrent infections, and anemia. To investigate whether this suppression of normal hematopoiesis is caused by spatial competition between normal hematopoietic stem cells and leukemic cells, Prim and Van Bekkum (1981) injected tritiated thymidine-labeled leukemic cells from the in vivo growing Brown Norway acute myelocytic leukemia (BNML) cell line, a wellcharacterized model of human AML (reviewed by Martens et al., 1990). into rats. It was subsequently observed that these cells were preferentially localized in the subendosteal region of the femoral bone marrow, in contrast to L4415 cells, a rat model for human acute lymphocytic leukemia (Kloosterman et al., 1992;Prins and Van Bekkum, 1981). This led to the assumption that AML cells specifically compete for space in the bone marrow compartment that is normally occupied by immature hemopoietic progenitor cells (Van Bekkum et al., 1981). To extend the study of the growth pattern and localization of acute myelocytic leukemia cells and to elucidate the role of adhesion molecules in this process, a more versatile and permanent method to mark leukemic cells was required.Genetic marking using the ErGherzchia coli B-galactosidase gene (LacZ) is a widely used method for the identification and localization of transplanted cells in vivo (Cui et al., 1994 an in vitro growing derivative of the BNML cell line (Lacaze et al., 1983). This resulted in the development of a genetically marked leukemic cell line, LT12NL15, which exhibits stable expression of large amounts of E. cofi P-galactosidase in the cytoplasm of every cell (Hendrikx et al., 1995;Yan et al., 1993). These cells were used to set up a sensitive system to study homing and growth of leukemic cells (Hendrikx et al., 1995). Here we report the development of an immunohistochemical staining method for Lac2 in paraffin sections of formalin-fixed, decalcified tibiae and femora of rats carrying LT12NL15 leukemia. This method allows detailed visualization of genetically marked leukemic cells within the undisturbed spatial context of the bone marrow and the enclosing bone. This procedure may also be of value to other studies in which detection of Lac2 labeled cells in p d i sections can o&r increased specificity, greater convenience, and superior tissue preservation compared to frozen sections. Materials and MethodsCells. LT12 is an in vitro as well as in vivo growing rat acute promyelocytic leukemia cell line, derived from the in vivo growing BNML cell line (Hagenbeek, 1977). LT12 cells were genetically marked using a retroviral vector containing the L a d gene, resulting in the subline LT12NL15. Briefl...