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Biomedical subjects

Leroy F Liu

Publications and source records attributed to Leroy F Liu.

At least 19 recordsLinked to original sources

Autophagy regulates ageing in C. elegans.

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.

Aging↗

G-quadruplexes induce apoptosis in tumor cells.

Several G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture. Systematic mutational analysis of the TG-ODNs suggests that the antiproliferative activity depends on the G-quadruplex conformation of these TG-ODNs. TG-ODNs were also shown to induce poly(ADP-ribose) polymerase-1 cleavage, phosphatidylserine flipping, and caspase activation, indicative of induction of apoptosis. TG-ODN-induced apoptosis was largely ataxia telangiectasia mutated (ATM) dependent. Furthermore, TG-ODN-induced apoptosis was inhibited by the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125. Indeed, TG-ODNs were shown to activate the JNK pathway in an ATM-dependent manner as evidenced by elevated phosphorylation of JNK and c-Jun. Interestingly, a number of G-quadruplex ODNs (GQ-ODN) derived from nontelomeric sequences also induced ATM/JNK-dependent apoptosis, suggesting a possible common mechanism of tumor cell killing by GQ-ODNs.

Animals↗

Role of apoptotic nuclease caspase-activated DNase in etoposide-induced treatment-related acute myelogenous leukemia.

Etoposide-induced treatment-related acute myelogenous leukemia (t-AML) is characterized by rearrangements of the mixed lineage leukemia (MLL) gene with one of its >50 partner genes, most probably as a consequence of etoposide-induced DNA double-strand breaks (DSBs). Recent studies have shown that etoposide-induced DSBs occur predominantly within the breakpoint cluster region (bcr) of the MLL gene. However, bcr-specific DSBs induced by etoposide are not topoisomerase II-linked but the result of apoptotic nuclease-mediated DNA cleavage. Here, we test the involvement of caspase-activated DNase (CAD) and other apoptotic components in etoposide-induced gene rearrangements using two methods. First, we measured the effect of etoposide on the integration frequency of a transfected plasmid. Etoposide strongly stimulated plasmid integration in CAD cDNA-complemented mouse embryonic fibroblasts (MEFs) but not in CAD knockout (KO) MEFs. Consistently, down-regulation of ICAD (inhibitor of CAD, also required for proper folding of CAD) in an HT29-derived cell line, which leads to decreased CAD activity, significantly reduced etoposide-induced plasmid integration. Second, we used long-template inverse PCR to focus on gene rearrangements at the MLL locus. Etoposide stimulated MLL fusion product formation in CAD cDNA-complemented MEFs but not in CAD KO MEFs. Together, these results suggest that CAD and other apoptotic components may play an important role in etoposide-induced t-AML.

Animals↗

A protease pathway for the repair of topoisomerase II-DNA covalent complexes.

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.

Animals↗

Role of topoisomerase IIbeta in the expression of developmentally regulated genes.

Mice lacking topoisomerase IIbeta (TopIIbeta) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2beta knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2beta knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIbeta is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIbeta and histone deacetylase 2, a known TopIIbeta-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIbeta binding to the 5' region of a number of TopIIbeta-sensitive genes. Further studies of a TopIIbeta-sensitive gene, Kcnd2, revealed the presence of TopIIbeta in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIbeta in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.

Animals↗

Potent antitumor 9-anilinoacridines and acridines bearing an alkylating N-mustard residue on the acridine chromophore: synthesis and biological activity.

A series of 9-anilinoacridine and acridine derivatives bearing an alkylating N-mustard residue at C4 of the acridine chromophore were synthesized. The N-mustard pharmacophore was linked to the C4 of the acridine ring with an O-ethyl (O-C(2)), O-propyl (O-C(3)), or O-butyl (O-C(4)) spacer. It revealed that all newly synthesized compounds were very potent cytotoxic agents against human leukemia and various solid tumors in vitro. These agents did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or taxol-resistant (CCRF-CEM/taxol) cells. It also showed that these agents were DNA cross-linking agents rather than topoisomerase II inhibitors. Of these agents, compounds 27a and 27c were shown to have potent antitumor activity in nude mice bearing the human breast carcinoma MX-1 xenograft. The therapeutic efficacies of these two agents are comparable to that of taxol.

Acridines↗

Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro.

Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.

Animals↗

6-Substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones: reversed lactam analogues of ARC-111 with potent topoisomerase I-targeting activity and cytotoxicity.

6-Substituted 8,9-dimethoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-ones were synthesized and evaluated for topoisomerase I-targeting activity and cytotoxicity. Several of these reversed lactam analogues of ARC-111 exhibited exceptional cytotoxicity with IC50 values ranging from 0.5 to 3.0 nM. In contrast to topotecan, no resistance was observed with several of these reversed lactam analogues in tumor cell lines that overexpressed the efflux transporters MDR1 or BCRP.

Amides↗

Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway.

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.

Breast Neoplasms↗

Esters and amides of 2,3-dimethoxy-8,9-methylenedioxy-benzo[i]phenanthridine-12-carboxylic acid: potent cytotoxic and topoisomerase I-targeting agents.

The exceptional topoisomerase I-targeting activity and antitumor activity of 5-(2-N,N-dimethylamino)ethyl-8,9-dimethoxy-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (ARC-111, topovale) prompted studies on similarly substituted benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. Among the benzo[i]phenanthridine-12-carboxylic esters evaluated, the 2-(N,N-dimethylamino)ethyl, 2-(N,N-dimethylamino)-1-methylethyl, and 2-(N,N-dimethylamino)-1,1-dimethylethyl esters possessed similar cytotoxicity, ranging from 30 to 55 nM in RPMI8402 and KB3-1 cells. Several of the carboxamide derivatives possess potent topoisomerase I-targeting activity and cytotoxicity. The 2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl, and 2-(pyrrolidin-1-yl)ethyl amides were among the more cytotoxic benzo[i]phenanthridine-12-carboxylic derivatives, with IC50 values ranging from 0.4 to 5.0 nM in RPMI8402 and KB3-1 cells.

Amides↗

Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: synthesis and biological activity.

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene (O-C(2)), O-butylene (O-C(4)), and methylene (C(1)) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting human lymphoblastic leukemic cells (CCRF-CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis (2-chloroethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lymphoblastic leukemic cells (CCRF-CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or Taxol-resistant (CCRF-CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the Taxol therapeutic dose [i.e., 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5); intravenous injection] on nude mice bearing human breast carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft tumor suppression of 81-96% using human T-cell acute lymphoblastic leukemia CCRF-CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma SK-OV-3 tumor models.

Alkylating Agents↗

5-(2-aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: variation of n-alkyl substituents modulates sensitivity to efflux transporters associated with multidrug resistance.

5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one (ARC-111) has potent TOP1-targeting activity and pronounced antitumor activity. Several analogues of ARC-111 were synthesized with NH2, N-alkyl, N,N-dialkyl, pyrrolidinyl, piperidinyl, and piperazinyl substituents at the 2-position of the 5-ethyl group. The relative TOP1-targeting activity and cytotoxicity of these structural analogues were assessed in RPMI8402 and P388 tumor cells and their camptothecin-resistant variants CPT-K5 and P388/CPT45, respectively. Potent TOP1-targeting activity was retained within a series of mono N-alkyl analogues that included NHCH2CH3, NHCH(CH3)2, and NHC(CH3)3. TOP1-targeting activity was diminished by the presence of a N-benzyl moiety. In a comparison of a series of N-alkyl-N-isopropyl analogues, activity decreased in the order CH3 > CH2CH3 > CH(CH3)2. Cytotoxicity in RPMI8402 and P388 did correlate with TOP1-targeting activity. Cytotoxic activity was also determined in KB3-1 cells and its variants KB/V-1 and KBH5.0. As KB/V-1 cells overexpress MDR1 and KBH5.0 cells overexpress BCRP, decreased cytotoxicity in these cell lines relative to the parent cell line is indicative of compounds that are substrates for these efflux transporters. In view of their diminished cytotoxicity in KB/V-1 cells, it appears that the likely demethylated metabolites of ARC-111, i.e., where NH2 or NHCH3 replaces the N(CH3)2 at the 2-position of the 5-ethyl substituent, are substrates for MDR1. In contrast, no significant difference in cytotoxicity among these three cell lines was observed with other N-alkyl analogues, including NHC2H5, NHCH(CH3)2, NHC(CH3)3, N(CH3)2, N(CH2CH3)2, NCH3(CH(CH3)2), and either the pyrrolidinyl or the piperidinyl analogues. The 2-(piperazinyl) analogues were associated with diminished cytotoxicity in KB/V-1 cells, suggesting that the second basic amino substituent is associated with their recognition as substrates by MDR1. Comparative studies on the antitumor activity of ARC-111 and its N-demethylated derivatives (the NHCH3 and NH2 analogues) against SJ-BT45 medulloblastoma xenografts in scid mice revealed that the secondary amine metabolite is at least as active as ARC-111 in vivo, although the primary amine derivative was significantly less potent.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Dimethoxybenzo[i]phenanthridine-12-carboxylic acid derivatives and 6H-dibenzo[c,h][2,6]naphthyridin-5-ones with potent topoisomerase I-targeting activity and cytotoxicity.

The exceptional TOP1-targeting activity and antitumor activity of ARC-111, 1, prompted studies on similarly substituted benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. These studies were extended to include 6-substituted 8,9-dimethoxy-2,3-methylenedioxy-dibenzo[c,h][2,6]naphthyridin-5-ones, which represent reversed lactam analogues of 1. Several of these analogues retained the potent TOP1-targeting activity and cytotoxicity observed for ARC-111.

Antineoplastic Agents↗

Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53.

The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.

Amino Acid Sequence↗

Nitro and amino substitution within the A-ring of 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: influence on topoisomerase I-targeting activity and cytotoxicity.

Recently, 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one, 1, was identified as a TOP1-targeting agent with pronounced antitumor activity. In the present study, the effect on activity of substituting a single nitro or amino group in the A-ring in lieu of the methylenedioxy moiety of 1 was evaluated. The presence of either a nitro or amino substituent at the 4-position had a pronounced adverse affect on both TOP1-targeting activity and cytotoxicity. To a lesser extent, derivatives with a nitro or amino substituent at the 1-position were also less active than 1. Replacement of the methylenedioxy moiety of 1 with either a nitro or amino substituent at either the 2- and 3-position did result in analogues with potent TOP1-targeting activity and cytotoxicity.

Amination↗

Nuclear translocation of cytochrome c during apoptosis.

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.

Active Transport, Cell Nucleus↗

11H-Isoquino[4,3-c]cinnolin-12-ones; novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity.

Recent studies have identified 2,3-dimethoxy-8,9-methylenedioxy-11-[(2-dimethylamino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1a) as a novel topoisomerase I-targeting agent with potent cytotoxic activity. The effect of varied substituents at the 11-position of 2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones on topoisomerase I-targeting activity and cytotoxicity was evaluated. Potent TOP1-targeting activity was observed when the 11-position was substituted with either a 2-(N,N-dimethylamino)ethyl, a 2-(N,N-diethylamino)ethyl, a n-butyl, or a 2-(pyrrolidin-1-yl)ethyl group. The addition of a beta-methyl group to 1a provided an analogue with dramatically reduced TOP1-targeting activity and cytotoxicity. Analogues of 1a wherein the 2-(N,N-dimethylamino)ethyl group was replaced with a (2-tetrahydrofuranyl)methyl, a 2-(piperidin-1-yl)ethyl, or a 2-(4-methylpiperazin-1-yl)ethyl substituent exhibited decreased activity as TOP1-targeting agents. Replacement of the dimethoxy groups of 1a with hydrogen atoms resulted in an analogue with significantly decreased TOP1-targeting activity and cytotoxicity. Removal of both the vicinal dimethoxyl groups and the methylenedioxy moiety resulted in a complete loss of TOP1-targeting activity. The presence of a 9-nitro substituent in place of the 8,9-methylenedioxy group of 1a resulted in a decrease in relative TOP1-targeting activity and cytotoxicity. Compounds 1a and the 11-n-butyl analogue 1d were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line MDA-MB-435 was used in these assays. At dose levels that approached its maximum tolerated dose, 1a proved to be effective in inhibiting tumor growth in vivo when administered orally or by ip injection.

Animals↗

Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death.

Reactive oxygen species modify DNA, generating various DNA lesions including modified bases such as 8-oxoguanine (8-oxoG). These base-modified DNA lesions have been shown to trap DNA topoisomerase I (TOP1) into covalent cleavage complexes. In this study, we have investigated the role of TOP1 in hydrogen peroxide toxicity. We showed that ectopic expression of TOP1 in Saccharomyces cerevisiae conferred sensitivity to hydrogen peroxide, and this sensitivity was dependent on RAD9 checkpoint function. Moreover, in the mammalian cell culture system, hydrogen peroxide-induced growth inhibition and apoptosis were shown to be partly TOP1-dependent as evidenced by a specific increase in resistance to hydrogen peroxide in TOP1-deficient P388/CPT45 murine leukemia cells as compared with their TOP1-proficient parental cell line P388. In addition, hydrogen peroxide was shown to induce TOP1-DNA cross-links. These results support a model in which hydrogen peroxide promotes the trapping of TOP1 on oxidative DNA lesions to form TOP1-DNA cleavage complexes that contribute to hydrogen peroxide toxicity.

Animals↗