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ABSTRACT: Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients
PLoS.ONE. 01/2011; 6(1).
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Cancer Research - CANCER RES. 01/2007; 67(18):8839-8846.
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ABSTRACT: IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/degradation. (Cancer Res 2006; 66(2): 921-8)
Cancer Res. 01/2006; 66(2).
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ABSTRACT: DNA topoisomerase II (TOP2) cleavable complexes represent an unusual type of DNA damage characterized by reversible TOP2-DNA cross-links and DNA double strand breaks. Many antitumor drugs and physiological stresses are known to induce TOP2 cleavable complexes leading to apoptotic cell death and genomic instability. However, the molecular mechanism(s) for repair of TOP2 cleavable complexes remains unclear. In the current studies, we show that TOP2 cleavable complexes induced by the prototypic TOP2 poison VM-26 are proteolytically degraded by the ubiquitin/26 S proteasome pathway. Surprisingly the TOP2beta isozyme is preferentially degraded over TOP2alpha isozyme. In addition, transcription inhibitors such as 5,6-dichlorobenzimidazole riboside and camptothecin can substantially block VM-26-induced TOP2beta degradation. These results are consistent with a model in which the repair of TOP2beta cleavable complexes may involve transcription-dependent proteolysis of TOP2beta to reveal the protein-concealed double strand breaks.
Journal of Biological Chemistry 12/2001; 276(44):40652-8. · 4.77 Impact Factor
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ABSTRACT: VP-16 (etoposide) has recently been shown to induce topoisomerase II (TOP2)-mediated DNA cleavage within the mixed lineage leukemia (MLL) breakpoint cluster region (bcr), suggesting a role of TOP2 in MLL gene rearrangement. In our current studies, we have compared the induction of DNA cleavage within the MLL bcr in different cell lines after treatment with various anticancer drugs. All anticancer drugs tested including VP-16 (a TOP2-directed drug), camptothecin (a topoisomerase I-directed drug), 5-fluorouracil and methotrexate (antimetabolites), and vinblastine (a microtubule inhibitor) induced the same site-specific cleavage within the MLL bcr. This cleavage was shown to be nuclease-mediated but not TOP2-mediated by the following observations: 1) drug-induced cleavage within the MLL bcr was not protein-linked; 2) unlike TOP2-mediated cleavage, drug-induced DNA cleavage within the MLL bcr was kinetically slow and coincided with the formation of the apoptotic nucleosomal DNA ladder; 3) drug-induced cleavage within the MLL bcr was unaffected in cells with reduced nuclear TOP2; and 4) drug-induced cleavage within the MLL bcr was abolished by the caspase inhibitor, Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-FMK. The possibility that an apoptotic nuclease may be involved in cleavage of the MLL bcr and MLL gene translocation is discussed.
Journal of Biological Chemistry 09/2001; 276(34):31590-5. · 4.77 Impact Factor
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ABSTRACT: Camptothecin (CPT) induces down-regulation of topoisomerase I (TOP1) via an ubiquitin/26S proteasome pathway. Studies using a panel of breast and colorectal cancer cell lines as well as primary nontransformed and oncogene-transformed cells have demonstrated that CPT-induced down-regulation exhibits a high degree of heterogeneity. In general, nontransformed cells are much more proficient in CPT-induced TOP1 down-regulation than their transformed counterparts. Among the breast and colorectal cancer cell lines, there was a general correlation between the extent of CPT-induced TOP1 down-regulation and CPT resistance. The breast cancer cell line ZR-75-1, the most sensitive to CPT, was completely defective in CPT-induced TOP1 down-regulation, whereas the breast cancer cell line BT474, the least sensitive to CPT, exhibited effective CPT-induced TOP1 down-regulation. The 26S proteasome inhibitor MG132 was shown to inhibit CPT-induced down-regulation of TOP1 in BT474 cells and selectively sensitized BT474 but not ZR-75-1 cells to CPT-induced cytotoxicity and apoptosis. In the aggregate, these results suggest that CPT-induced down-regulation of TOP1 could be an important parameter for determining CPT sensitivity/resistance in tumor cells. Analysis of the levels of TOP1 cleavable complexes, SUMO-1-TOP1 conjugates, and ubiquitin-TOP1 conjugates in ZR-75-1 and BT474 cells has suggested that the heterogeneity of CPT-induced down-regulation of TOP1 in tumor cells is at least in part attributable to altered regulation of a process(es) downstream from the TOP1 cleavable complex.
Cancer Research 09/2001; 61(15):5926-32. · 7.86 Impact Factor
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ABSTRACT: Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.
Journal of Biological Chemistry 06/2001; 276(19):15990-5. · 4.77 Impact Factor
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ABSTRACT: The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.
Biochemistry 04/2001; 40(11):3316-23. · 3.42 Impact Factor
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ABSTRACT: DNA topoisomerases are double-edged swords. They are essential for many vital functions of DNA during normal cell growth. However, they are also highly vulnerable under various physiological and nonphysiological stresses because of their delicate act on breaking and rejoining DNA. These stresses (e.g. exposure to topoisomerase poisons, acidic pH, and oxidative stresses) can convert DNA topoisomerases into DNA-breaking nucleases, resulting in cell death and/or genomic instability. The importance of topoisomerase-mediated DNA cleavage in tumor cell death and carcinogenesis has been recognized. This review focuses on recent findings concerning the molecular mechanisms of the stress responses to topoisomerase-mediated DNA damage. The involvement of ubiquitin/26S proteasome and SUMO/UBC9 in these processes, as well as the role of topoisomerase cleavable complexes in apoptotic cell death are discussed.
Annual Review of Pharmacology 02/2001; 41:53-77. · 21.64 Impact Factor
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ABSTRACT: Topoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the processes of transcription, translation and mitosis. With the introduction of new clinical agents that are effective in poisoning topoisomerase I, this enzyme has proved to be an attractive molecular target in the development of anticancer drugs. Several terbenzimidazoles have been identified as potent topoisomerase I poisons. Structure-activity data on various terbenzimidazoles have revealed that the presence of lipophilic substituents at the 5-position of various terbenzimidazoles correlates with enhanced cytotoxicity. While the effect of having substituents at both the 5- and 6-positions had not been evaluated, previous studies did indicate that the presence of a fused benzo-ring at the 5,6-position results in a significant decrease in topoisomerase I poisoning activity and cytotoxicity. In the present study we investigated whether substituents at both the 5- and 6-positions of varied terbenzimidazoles would allow for retention of topo I poisoning activity. The 6-bromo, 6-methoxy, or 6-phenyl derivatives of both 5-bromo- and 5-phenylterbenzimidazole were synthesized and evaluated for topo I poisoning activity, as well as their cytotoxicity toward human lymphoblastoma cells. The data indicate that such derivatives do retain similar topo I poisoning activity and possess cytotoxicity equivalent to either 5-bromo- or 5-phenylterbenzimidazole. Significant enhancement in the topoisomerase I poisoning activity and cytotoxicity of 5-phenylterbenzimidazole is observed when the 2"-position is substituted with either a chloro or trifluoromethyl substituent. The influence of such substituents on the biological activity of 5.6-dibromoterbenzimidazole (6a) was also explored. In the case of either 2"-chloro-5,6-dibromoterbenzimidazole (6b) or 2"-trifluoromethyl-5,6-dibromoterbenzimidazole (6c), topoisomerase I poisoning was not enhanced relative to 6a. While cytotoxicity toward RPMI 8402 was also not significantly affected, comparative studies performed against several solid human tumor cell lines did reveal a significant increase in cytotoxicity observed for 6c as compared to 6a.
Bioorganic & Medicinal Chemistry 12/2000; 8(11):2591-600. · 2.92 Impact Factor
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ABSTRACT: Many DNA binding ligands (e.g., nogalamycin, actinomycin D, terbenzimidazoles, indolocarbazoles, nitidine, and coralyne) and various types of DNA lesions (e.g., UV dimers, DNA mismatches, and abasic sites) are known to stimulate topoisomerase I-mediated DNA cleavage. However, the mechanism(s) by which these covalent and noncovalent DNA interactions stimulate topoisomerase I-mediated DNA cleavage remains unclear. Using nogalamycin as a model, we have studied the mechanism of ligand-induced topoisomerase I-mediated DNA cleavage. We show by both mutational and DNA footprinting analyses that the binding of nogalamycin to an upstream site (from position -6 to -3) can induce highly specific topoisomerase I-mediated DNA cleavage. Substitution of this nogalamycin binding site with a DNA bending sequence (A(5)) stimulated topoisomerase I-mediated DNA at the same site in the absence of nogalamycin. Replacement of the A(5) sequence with a disrupted DNA bending sequence (A(2)TA(2)) significantly reduced the level of topoisomerase I-mediated DNA cleavage. These results, together with the known DNA bending property of nogalamycin, suggest that the nogalamycin-DNA complex may provide a DNA structural bend to stimulate topoisomerase I-mediated DNA cleavage.
Biochemistry 09/2000; 39(32):9928-34. · 3.42 Impact Factor
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ABSTRACT: Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.
Journal of Biological Chemistry 09/2000; 275(34):26066-73. · 4.77 Impact Factor
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ABSTRACT: 5-Phenylterbenzimidazole (1) is active as a topoisomerase I poison (topo I) and is cytotoxic to human tumor cells. No cross-resistance was observed for 1 when it was evaluated against the camptothecin-resistant cell line, CPT-K5. Derivatives of 1 substituted at the 2"-position, however, did exhibit cross-resistance to this cell line. The basis for the resistance of this cell line towards CPT is that it possesses a mutant form of topo I. These results suggest that substituents at the 2"-position may be in proximity to the wild-type enzyme. Therefore, we hypothesized that terbenzimidazoles with 2"-substituents could be capable of interacting with the enzyme and thereby influence activity within this class of topo I poisons. 5-Phenylterbenzimidazoles with a hydroxy, hydroxymethyl, mercapto, amino, N-benzoylaminomethyl, chloro, and trifluoromethyl group at the 2"-position were synthesized. In addition, several 2"-ethyl-5-phenylterbenzimidazoles were prepared containing either a methoxy, hydroxy, amino, or N-acetylamino group at the 2-position of the ethyl side-chain. These 2"-substituted 5-phenylterbenzimidazoles were evaluated as topo I poisons and for cytotoxic activity. The presence of a strong electron-withdrawing group at the 2"-position, such as a chloro or trifluoromethyl group, did enhance both topo I poisoning activity and cytotoxicity. Studies on the relative DNA binding affinity of 1 to its 2"-amino and 2"-trifluoromethyl derivatives did exhibit a correlation with their relative differences in biological activity.
Bioorganic & Medicinal Chemistry 07/2000; 8(6):1371-82. · 2.92 Impact Factor
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ABSTRACT: Coralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group, which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines studied, 5,6-dihydro-3,4-methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I poisoning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other protoberberine derivatives, these benz[a]acridine derivatives possess distinctly different physicochemical properties and represent a novel series of topo I poisons.
Bioorganic & Medicinal Chemistry 06/2000; 8(5):1171-82. · 2.92 Impact Factor
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ABSTRACT: Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I poisoning. We have employed a broad range of biophysical, biochemical, and computer modeling techniques to characterize and cross-correlate the DNA-binding and topoisomerase poisoning properties of four protoberberine analogues that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an observation consistent with an intercalative mode of interaction. (ii) Enthalpically favorable interactions, such as stacking interactions between the intercalated ligand and the neighboring base pairs, provide <50% of the thermodynamic driving force for the complexation of the protoberberines to duplex DNA. Computer modeling studies on protoberberine-DNA complexes suggest that only rings C and D intercalate into the host DNA helix, while rings A and B protrude out of the helix interior into the minor groove. (iii) All four protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of the D-ring influence both DNA binding and topoisomerase I poisoning properties. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoning activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines. (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a profound influence on topoisomerase I poisoning activity. Specifically, protoberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3, 4-methylenedioxy substituents all bind DNA with a similar affinity. By contrast, these analogues exhibit markedly different topoisomerase I poisoning activities, with these activities following the hierarchy: 3,4-methylenedioxy > 2,3-dimethoxy > 3, 4-dimethoxy. These differences in topoisomerase I poisoning activity may reflect the differing abilities of the analogues to interact with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consistent with a mechanistic model in which both ligand-DNA and ligand-enzyme interactions are important for the poisoning of topoisomerase I by protoberberines, with the DNA-directed interactions involving ring D and the enzyme-directed interactions involving ring A. It is reasonable to suggest that the poisoning of topoisomerase I by a broad range of other naturally occurring and synthetic ligands may entail a similar mechanism.
Biochemistry 06/2000; 39(24):7107-16. · 3.42 Impact Factor
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ABSTRACT: Ubiquitin/26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1/Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1/Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1/Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9/SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.
Proceedings of the National Academy of Sciences 05/2000; 97(8):4046-51. · 9.68 Impact Factor
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ABSTRACT: A series of 2'-heterocyclic derivatives of 5-phenyl-2,5'-1H-bibenzimidazoles were evaluated for topoisomerase I poisoning activity and cytotoxicity. Topo I poisoning activity was associated with 2'-derivatives that possessed a hydrogen atom capable of hydrogen bond formation, suggesting that the interatomic distances between such hydrogen atoms and the heteroatoms on the adjacent benzimidazole influence activity.
Bioorganic & Medicinal Chemistry Letters 05/2000; 10(8):719-23. · 2.55 Impact Factor
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ABSTRACT: Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo-I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S-phase-specific killing through potentially lethal collisions between advancing replication forks and topo-I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long-lived covalent topo-I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo-I-mediated DNA damage involving covalent modifications of topo-I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo-I (CPT-induced topo-I downregulation). The second involves SUMO conjugation to topo-I. The potentials roles of these new mechanisms for repair of topo-I-mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.
Annals of the New York Academy of Sciences 02/2000; 922:1-10. · 3.15 Impact Factor
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Annals of the New York Academy of Sciences 02/2000; 922:306-8. · 3.15 Impact Factor
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Annals of the New York Academy of Sciences 02/2000; 922:340-2. · 3.15 Impact Factor
