Wnt/ß-catenin signaling regulated SATB1 promotes colorectal cancer tumorigenesis and progression.

The chromatin organizer SATB1 has been implicated in the development and progression of multiple cancers including breast and colorectal cancers. However, the regulation and role of SATB1 in colorectal cancers is poorly understood. Here, we demonstrate that expression of SATB1 is induced upon hyperactivation of Wnt/ß-catenin signaling and repressed upon depletion of TCF7L2 (TCF4) and ß-catenin. Using several colorectal cancer cell line models and the APC min mutant zebrafish in vivo model, we established that SATB1 is a novel target of Wnt/ß-catenin signaling. We show that direct binding of TCF7L2/ß-catenin complex on Satb1 promoter is required for the regulation of SATB1. Moreover, SATB1 is sufficient to regulate the expression of ß-catenin, members of TCF family, multiple downstream effectors and mediators of Wnt pathway. SATB1 potentiates the cellular changes and expression of key cancer-associated genes in non-aggressive colorectal cells, promotes their aggressive phenotype and tumorigenesis in vivo. Conversely, depletion of SATB1 from aggressive cells reprograms the expression of cancer-associated genes, reverses their cancer phenotype and reduces the potential of these cells to develop tumors in vivo. We also show that SATB1 and ß-catenin bind to the promoters of TCF7L2 and the downstream targets of Wnt signaling and regulate their expression. Our findings suggest that SATB1 shares a feedback regulatory network with TCF7L2/ß-catenin signaling and is required for Wnt signaling-dependent regulation of ß-catenin. Collectively, these results provide unequivocal evidence to establish that SATB1 reprograms the expression of tumor growth- and metastasis-associated genes to promote tumorigenesis and functionally overlaps with Wnt signaling critical for colorectal cancer tumorigenesis.

Novel role of curcumin in the prevention of cytokine-induced islet death in vitro and diabetogenesis in vivo.

BACKGROUND AND PURPOSE: Oxidative stress caused by cytokine exposure is a major cause of pancreatic islet death in vitro and of diabetogenesis. Antioxidant compounds may prevent cytokine-induced damage to islet cells. Hence, we studied the potential of curcumin, an antioxidant and anti-inflammatory compound, in vitro to protect islets against pro-inflammatory cytokines and in vivo to prevent the progression of diabetes induced by multiple low doses of streptozotocin (MLD-STZ). EXPERIMENTAL APPROACH: Pancreatic islets from C57/BL6J mice were pretreated with curcumin (10 microM) and then exposed to a combination of cytokines. Islet viability, reactive oxygen species (ROS), NO, inducible NO synthase and NF-kappaB translocation were studied. Curcumin pretreated (7.5 mg kg(-1) day(-1)) C57/BL6J mice were given MLD-STZ (40 mg kg(-1)), and various parameters of diabetes induction and progression were monitored. KEY RESULTS: Curcumin protected islets from cytokine-induced islet death in vitro by scavenging ROS and normalized cytokine-induced NF-kappaB translocation by inhibiting phosphorylation of inhibitor of kappa B alpha (IkappaBalpha). In vivo, curcumin also prevented MLD-STZ, as revealed by sustained normoglycaemia, normal glucose clearance and maintained pancreatic GLUT2 levels. Pro-inflammatory cytokine concentrations in the serum and pancreas were raised in STZ-treated animals, but not in animals pretreated with curcumin before STZ. CONCLUSIONS AND IMPLICATIONS: Here, we have demonstrated for the first time that curcumin in vitro protects pancreatic islets against cytokine-induced death and dysfunction and in vivo prevents STZ-induced diabetes.

SATB1 cleavage by caspase 6 disrupts PDZ domain-mediated dimerization, causing detachment from chromatin early in T-cell apoptosis.

SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ~50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

Developmental and hormonal regulation of type II DNA topoisomerase in rat testis.

Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIalpha.

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIalpha to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIalpha. The results indicate that topo IIalpha binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIalpha displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIalpha to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIalpha. These results implicate a dual role for topo IIalpha in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.

Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase.

DNA topoisomerases are ubiquitous nuclear enzymes that govern the topological interconversions of DNA by transiently breaking/rejoining the phosphodiester backbone of one (type I) or both (type II) strands of the double helix. Consistent with these functions, topoisomerases play key roles in many aspects of DNA metabolism. Type II DNA topoisomerase (topo II) is vital for various nuclear processes, including DNA replication, chromosome segregation, and maintenance of chromosome structure. Topo II expression is regulated at multiple stages, including transcriptional, posttranscriptional, and posttranslational levels, by a multitude of signaling factors. Topo II is also the cellular target for a variety of clinically relevant anti-tumor drugs. Despite significant progress in our understanding of the role of topo II in diverse nuclear processes, several important aspects of topo II function, expression, and regulation are poorly understood. We have focused this review specifically on eukaryotic DNA topoisomerase II, with an emphasis on functional and regulatory characteristics.

Selective cleavage of BLM, the bloom syndrome protein, during apoptotic cell death.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.

Ku acts in a unique way at the mammalian telomere to prevent end joining.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Caught in the act: binding of Ku and PARP to MARs reveals novel aspects of their functional interaction.

Specific regions of eukaryotic genomic DNA that exhibit high-affinity binding to the nuclear matrix in vitro are called matrix attachment regions (MARs) and are implicated in the loop domain organization of chromatin. Small regions possessing high base unpairing potential within these MARs are referred to as base unpairing regions (BURs). BUR-affinity chromatographic separations of proteins from breast cancer cells yielded, almost exclusively, a mixture of poly (ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK), two nuclear enzymes that are implicated in the cellular response to DNA damage. Contrary to the long-held notion that PARP and Ku autoantigen, the DNA-binding heterodimeric subunit of DNA-PK, bind only to DNA ends, recently we have shown that both proteins individually bind BURs with high affinity and specificity in an end-independent manner. Furthermore, Ku autoantigen forms a molecular complex with PARP in the absence of DNA, and the physical association of these proteins synergistically enhanced their BUR-binding activity. Autoribosylation of PARP abolished its association with Ku autoantigen and BUR-binding activity. These findings have, for the first time, provided a molecular link toward elucidating the functional interaction between PARP and DNA-PK. The identification of MARs as their common binding target suggests a novel role for these enzymes in the modulation of chromatin structure and function.

Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences.

Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.

Effects of nucleosomes and anti-tumor drugs on the catalytic activity of type II DNA topoisomerase from rat testis.

To gain insight into the relative catalytic efficiencies of mammalian type I and type II DNA topoisomerases, in the cellular context, we have used naked DNA and DNA incorporated into nucleosomes as substrates. We observed that the relaxation activity of both the enzymes declined with DNA containing increasing densities of nucleosomes; however, kinetic analysis revealed that topoisomerase I seemed less affected than topoisomerase II. The addition of histone H1, in stoichiometric amounts, to naked DNA or minichromosomes lessened the activity of topoisomerase II, and required 7-fold less for complete inhibition when the latter was used as the substrate. To ascertain if the observed differences are specific to topoisomerase II from testis, we examined the effect of nucleosomes on the catalytic efficiency of its isoform from liver. Interestingly, the suppression of relaxation activity of liver topoisomerase II required substrates containing higher mass ratios of histone octamer/DNA. Studies on the effect of nucleosomes on the action of teniposide displayed significant differences in the kinetics of the reaction, in its IC50 values, and have provided biochemical evidence for the first time that nucleosomes increased inhibition caused by teniposide. Further, this feature appears to be specific for topoisomerase II-directed drugs and is not shared by the generic class of either DNA-intercalating or non-DNA-intercalating ligands.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver.

A number of studies in yeast have shown that DNA topoisomerase II is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase II in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-free extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg(2+)-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.

Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses.

Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.