Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture.
2018; 175 (6): 1607–19.e15
Telomeric 3 ' Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST
2012; 150 (1): 39-52
In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.
View details for DOI 10.1016/j.cell.2018.11.012
View details for PubMedID 30500539
Apollo Contributes to G Overhang Maintenance and Protects Leading-End Telomeres
2010; 39 (4): 606-617
A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.
View details for DOI 10.1016/j.cell.2012.05.026
View details for Web of Science ID 000306115000005
View details for PubMedID 22748632
View details for PubMedCentralID PMC3392515
Human Telomerase caught in the Act
2009; 138 (3): 432-434
Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.
View details for DOI 10.1016/j.molcel.2010.06.031
View details for Web of Science ID 000281421100013
View details for PubMedID 20619712
View details for PubMedCentralID PMC2929323
No overt nucleosome eviction at deprotected telomeres
MOLECULAR AND CELLULAR BIOLOGY
2008; 28 (18): 5724-5735
Based on prior work, it was expected that telomerase would preferentially elongate the shortest telomeres in a cell, extending the telomeric G-rich strand through a process that is coupled to the synthesis of the complementary strand. Contrary to this view, Zhao et al. (2009) now show that telomerase in human cancer cells extends most telomeres during every S phase and that complementary strand synthesis does not immediately follow telomerase action.
View details for DOI 10.1016/j.cell.2009.07.018
View details for Web of Science ID 000268771900009
View details for PubMedID 19665967
Assessment of antiangiogenic effect using 99mTc-EC-endostatin.
Cancer biotherapy & radiopharmaceuticals
2002; 17 (2): 233–45
Dysfunctional telomeres elicit the canonical DNA damage response, which includes the activation of the ATM or ATR kinase signaling pathways and end processing by nonhomologous end joining (NHEJ) or homologous recombination (HR). The cellular response to DNA double-strand breaks has been proposed to involve chromatin remodeling and nucleosome eviction, but whether dysfunctional telomeres undergo chromatin reorganization is not known. Here, we report on the nucleosomal organization of telomeres that have become deprotected through the deletion of the shelterin components TRF2 or POT1. We found no evidence of changes in the nucleosomal organization of the telomeric chromatin or nucleosome eviction near the telomere terminus. An unaltered chromatin structure was observed at telomeres lacking TRF2, which activate the ATM kinase and are a substrate for NHEJ. Similarly, telomeres lacking POT1a and POT1b, which activate the ATR kinase, showed no overt nucleosome eviction. Finally, telomeres lacking TRF2 and Ku70, which are processed by HR, appeared to maintain their original nucleosomal organization. We conclude that ATM signaling, ATR signaling, NHEJ, and HR at deprotected telomeres can take place in the absence of overt nucleosome eviction.
View details for DOI 10.1128/MCB.01764-07
View details for Web of Science ID 000258951000014
View details for PubMedID 18625717
View details for PubMedCentralID PMC2546919
In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTc-EC-annexin V.
Cancer biotherapy & radiopharmaceuticals
2001; 16 (1): 73–83
Tumor vascular density may provide a prognostic indicator of metastatic potential or survival. The purpose of this study was to develop 99mTc-ethylenedicysteine-endostatin (99mTc-EC-endostatin) for the evaluation of anti-angiogenesis therapy.99mTc-EC-endostatin was prepared by conjugating ethylenedicysteine (EC) to endostatin, followed by adding pertechnetate and tin chloride. Radiochemical purity was > 95%. In vitro cell viability, affinity and TUNEL assays were performed. Tissue distribution and planar imaging of radiolabeled endostatin were determined in tumor-bearing rats. To assess anti-angiogenic treatment response, rats were treated with endostatin, paclitaxel and saline, followed by imaging with 99mTc-EC-endostatin. Tumor response to endostatin therapy in tumor-bearing animal models was assessed by correlating tumor uptake dose with microvessel density, VEGF, bFGF and IL-8 expression during endostatin therapy.In vitro cell viability and TUNEL assays indicated no marked difference between EC-endostatin and endostatin. Cellular uptake assay suggests that endostatin binds to endostatin receptor. Biodistribution of 99mTc-EC-endostatin in tumor-bearing rats showed increased tumor-to-tissue count density ratios as a function of time. Tumor uptake (%ID/g) of 99mTc-EC-endostatin was 0.2-0.5. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-endostatin. The optimal time for imaging using radiolabeled endostatin was 2 hrs. 99mTc-EC-endostatin could assess treatment response. There was a correlation between tumor uptake and cellular targets expression.The results indicate that it is feasible to use 99mTc-EC-endostatin to assess efficiency of anti-angiogenesis therapy.
View details for DOI 10.1089/108497802753773856
View details for PubMedID 12030117
The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using 99mTc-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression.Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and day 5 post-treatment.In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with 99mTc-EC in the same time period. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection.The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.
View details for DOI 10.1089/108497801750096087
View details for PubMedID 11279800