We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

Cancer and many other diseases are characterized by changes in cell morphology, motion, and mechanical rigidity. However, in live cell cytology, stimulus-induced morphologic changes typically take 10-30 min to detect. Here, we employ live-cell interferometry (LCI) to visualize the rapid response of a whole cell to mechanical stimulation, on a time scale of seconds, and we detect cytoskeletal remodeling behavior within 200 s. This behavior involved small, rapid changes in cell content and miniscule changes in shape; it would be difficult to detect with conventional or phase contrast microscopy alone and is beyond the dynamic capability of AFM. We demonstrate that LCI provides a rapid, quantitative reconstruction of the cell body with no labeling. This is an advantage over traditional microscopy and flow cytometry, which require cell surface tagging and/or destructive cell fixation for labeling.

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The dynamic nanomechanical properties of a large number of cells (up to hundreds), measured in parallel with high throughput, are reported. Using NIH 3T3 and HEK 293T fibroblasts and actin depolymerizing drugs, we use a novel nanotechnology to quantify the local viscoelastic properties with applied forces of 20 pN-20 nN, a spatial resolution of <20 nm, and a mechanical dynamic range of several Pa up to ~200 kPa. Our approach utilizes imaging interferometry in combination with reflective, magnetic probes attached to cells. These results indicate that mechanical imaging interferometry is a sensitive and scalable technology for measuring the nanomechanical properties of large arrays of live cells in fluid.