Feridoun Karimi-Busheri

XRCC1 Stimulates Human Polynucleotide Kinase Activity at Damaged DNA Termini and Accelerates DNA Single-Strand Break Repair

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining

The efficient repair of double‐strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non‐homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non‐ conventional end groups that must be processed to restore the ligatable 3′‐OH and 5′‐phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell‐free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5′‐OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA‐PKcs, indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end‐joining activity can be restored to PNK‐depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.

Molecular characterization of a human DNA kinase.

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5′-hydroxyl termini and dephosphorylate 3′-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5′-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes ofCaenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.

Senescence evasion by MCF-7 human breast tumor-initiating cells

IntroductionA subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence.MethodsTo test this hypothesis, we isolated CD24-/low/CD44+ tumor-initiating cells (as mammospheres) from MCF-7 breast cancer cells grown in adherent monolayer culture, and carried out a comprehensive comparison of cell death and DNA damage response pathways prior to and after exposure to ionizing radiation in mammospheres and monolayer MCF-7 cells. Single and double-strand break repair was measured by single-cell gel electrophoresis. The latter was also examined by phosphorylation of histone H2AX and formation of 53BP1 and Rad51 foci. Apoptosis was quantified by flow-cytometric analysis of annexin V-binding and senescence was analyzed on the basis of cellular β-galactosidase activity. We employed the telomeric repeat amplification protocol to quantify telomerase activity. Expression of key DNA repair and cell cycle regulatory proteins was detected and quantified by western blot analysis.ResultsOur data demonstrate that in comparison to the bulk population of MCF-7 cells (predominantly CD24+/CD44+), the MCF-7 mammosphere cells benefit from a multifaceted approach to cellular protection relative to that seen in monolayer cells, including a reduced level of reactive oxygen species, a more active DNA single-strand break repair (SSBR) pathway, possibly due to a higher level of expression of the key SSBR protein, human AP endonuclease 1 (Ape1), and a significantly reduced propensity to undergo senescence as a result of increased telomerase activity and a low level of p21 protein expression. No significant difference was seen in the rates of double-strand break repair (DSBR) between the two cell types, but DSBR in mammospheres appears to by-pass the need for H2AX phosphorylation.ConclusionsEnhanced survival of MCF-7 tumor-initiating cells in response to ionizing radiation is primarily dependent on an inherent down-regulation of the senescence pathway. Since MCF-7 cells are representative of cancer cells that do not readily undergo apoptosis, consideration of senescence pathways may play a role in targeting stem cells from such tumors.

PATHWAYS OF PROLIFERATION AND ANTIAPOPTOSIS DRIVEN IN BREAST CANCER STEM CELLS BY STEM CELL PROTEIN PIWIL2

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X(L) and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells.

Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5-DNA kinase activity and a 3-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5-phosphate and 3-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 dead-end complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.

XRCC1 stimulates polynucleotide kinase by enhancing its damage discrimination and displacement from DNA repair intermediates

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5′-DNA kinase and 3′-phosphatase activities of hPNK can be stimulated by the “scaffold” protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5′-OH termini was only ∼5-fold tighter than that to identical DNA molecules with 5′-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5′-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5′-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5′-OH termini and those with 5′-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5-termini and dephosphorylates DNA 3-termini. hPNKP is involved in both single- and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to gamma-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC50 of 0.06 micromol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 micromol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor beta1.

Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi‐layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor‐β1 (TGF‐β1), it is not clear whether differentiated keratinocytes in a multi‐layer form release this multi‐functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono‐ and multi‐layer forms express different levels of TGF‐β1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 μg/ml) and EGF (5 μg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi‐layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF‐β1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and 3H‐thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF‐β1, involucrin, TIMP‐1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1–2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF‐β1 evaluation revealed that mono‐layers of cultured keratinocytes which were round, attached, and proliferating in KSFMu2009+u2009BPE and EGF containing medium released a significantly higher level of TGF‐β1 (196u2009±u200958 pg /ml) relative to those grown in multi‐layer forms (28u2009±u20097.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF‐β1 (388u2009±u200953 pg/ml) relative to those grown in KSFMu2009+u2009BPE and EGF. This finding was consistent with the expression of TGF‐β1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF‐β1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of 3H‐thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF‐β1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF‐β1 and a fibroblast mitogenic factor. J. Cell. Biochem. 83: 239–248, 2001.

Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver

Damage to DNA can result in strand breaks with 5′‐hydroxyl and 3′‐phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5′‐phosphate and 3′‐hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS‐polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are ∼60‐kDa polypeptides. Both enzymes have an acidic pH optimum (5.5–6.0) for kinase activity, and similar pl values (8.5–8.6), and a specificity for DNA. The calf thymus kinase possesses a 3′‐phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7–8 nucleotides in length, but the optimal size appears to be >18 nucleotides. Comparison of phosphorylation of oligo(dA)24 and oligo(dT)24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5′‐overhanging termini when acting at DNA termini produced by restriction enzymes. With double‐stranded oligonucleotide complexes designed to model single‐strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5′‐terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single‐strand breaks. J. Cell. Biochem. 64:258–272.