Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G 2 phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.

Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key CSR player, targets IgH switch (S) regions, which contain 5′-AGCT-3′ repeats in their core. How AID is recruited to S regions remains unclear. Here we show that 14-3-3 adaptor proteins play an important role in CSR. 14-3-3 proteins specifically bind 5′-AGCT-3′ repeats, are upregulated in B cells undergoing CSR and are recruited together with AID to the S regions involved in CSR events (Sμ→Sγ1, Sμ→Sγ3 or Sμ→Sα). Moreover, blocking 14-3-3 by difopein, deficiency in 14-3-3γ or expression of a dominant negative 14-3-3σ mutant impaired recruitment of AID to S regions and decreased CSR. Finally, 14-3-3 proteins interact directly with AID and enhance AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.

Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI 50 , 10-21 Mmol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds. [Cancer Res 2008;68(20):8393-9]

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.

Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole-or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules.