Showing papers by "Eppley Institute for Research in Cancer and Allied Diseases published in 2006"
TL;DR: The cytoplasmic tail of MUC1 (MUC1CT), the best characterized of the transmembrane mucins, is involved in several signaling pathways, including those involving Ras, beta-catenin, p120 caten in, p53 and estrogen receptor alpha.
385 citations
TL;DR: Evidence strongly indicates that estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts, the first in vitro/in vivo model of estrogen-induced carcinogenesis in human breast epithelial cells.
381 citations
TL;DR: New procedures for derivation and maturation of these stem cells into the tissue‐specific cell progenitors are described, with emphasis on the interactions among certain developmental signaling factors, such as hormones, epidermal growth factor, hedgehog, Wnt/β‐catenin, and Notch.
Abstract: In this study, we report on recent advances on the functions of embryonic, fetal, and adult stem cell progenitors for tissue regeneration and cancer therapies. We describe new procedures for derivation and maturation of these stem cells into the tissue-specific cell progenitors. The localization of the adult stem cells and their niches, as well as their implication in the tissue repair after injuries and during cancer progression, are also described. The emphasis is on the interactions among certain developmental signaling factors, such as hormones, epidermal growth factor, hedgehog, Wnt/beta-catenin, and Notch. These factors and their pathways are involved in the stringent regulation of the self-renewal and/or differentiation of adult stem cells. Novel strategies for the treatment of both diverse degenerating disorders, by cell replacement, and some metastatic cancer types, by molecular targeting multiple tumorigenic signaling elements in cancer progenitor cells, are also illustrated.
289 citations
TL;DR: Some physiologically relevant regulatory aspects of mucin production are discussed, with a particular emphasis on aberrations that pertain to pathological situations.
287 citations
TL;DR: The relative reactivities of E2-3,4-Q and E-2-2,3-Q to form depurinating adducts correlate with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-OHE2 and 2- OHE2.
Abstract: Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of ∼3 h at 37 °C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5−20 h at 37 °C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 °C. Depurinating and stable adducts were analyzed. In similar ...
163 citations
TL;DR: The structure, the mechanism, and the evolutionary relationships of DNA polymerases and their possible functions in the replication of intact and damaged chromosomes, DNA damage repair, and recombination are discussed.
Abstract: The functioning of the eukaryotic genome depends on efficient and accurate DNA replication and repair. The process of replication is complicated by the ongoing decomposition of DNA and damage of the genome by endogenous and exogenous factors. DNA damage can alter base coding potential resulting in mutations, or block DNA replication, which can lead to double-strand breaks (DSB) and to subsequent chromosome loss. Replication is coordinated with DNA repair systems that operate in cells to remove or tolerate DNA lesions. DNA polymerases can serve as sensors in the cell cycle checkpoint pathways that delay cell division until damaged DNA is repaired and replication is completed. Eukaryotic DNA template-dependent DNA polymerases have different properties adapted to perform an amazingly wide spectrum of DNA transactions. In this review, we discuss the structure, the mechanism, and the evolutionary relationships of DNA polymerases and their possible functions in the replication of intact and damaged chromosomes, DNA damage repair, and recombination.
126 citations
TL;DR: The hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of M UC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma is supported.
Abstract: MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.
119 citations
Journal Article•
TL;DR: Although I3C has been shown to protect against tumor induction by some carcinogens, it has also been observed to promote tumor development in animal models and to increase the formation of estrogen metabolites that induce or promote cancer, but this has not been demonstrated.
Abstract: The hydrolysis product of glucobrassicin, indole-3-carbinol (I3C), is metabolized to a variety of products, including the dimeric 3,3'-diindolylmethane (DIM). Both I3C and DIM exert a variety of biological and biochemical effects. Most of these effects appear to occur because I3C modulates several nuclear transcription factors. I3C induces phase I and phase II enzymes that metabolize carcinogens, including estrogens. Administration of either I3C or DIM results in increased 2-hydroxylation of estrogens. I3C also enhances DNA repair by affecting several of the proteins involved in this process. I3C induces both G1 cell cycle arrest and apoptosis. All of these activities lead to anticancer effects. Although I3C has been shown to protect against tumor induction by some carcinogens, it has also been observed to promote tumor development in animal models. In humans, I3C and DIM affect the metabolism of estrogens. Concerns have been raised that I3C might increase the formation of estrogen metabolites that induce or promote cancer, but this has not been demonstrated. I3C has been found to be effective in treating some cases of recurrent respiratory papillomatosis, and it may have other clinical uses.
112 citations
TL;DR: This unifying mechanism of the origin of cancer and other diseases suggests preventive strategies based on the level of depurinating DNA adducts that generate the first critical step in the initiation of diseases.
Abstract: Estrogens can be converted to electrophilic metabolites, particularly the catechol estrogen-3,4-quinones, estrone(estradiol)-3,4-quinone [E(1)(E(2))-3,4-Q], which react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone repair of this damage leads to the mutations that initiate breast, prostate, and other types of cancer. The reaction of E(1)(E(2))-3,4-Q with DNA forms the depurinating adducts 4-hydroxyE(1)(E(2))-1-N3adenine [4-OHE(1)(E(2))-1-N3Ade] and 4-OHE(1)(E(2))-1-N7guanine(Gua). These two adducts constitute >99% of the total DNA adducts formed. The E(1)(E(2))-2,3-Q forms small amounts of the depurinating 2-OHE(1)(E(2))-6-N3Ade adducts. Reaction of the quinones with DNA occurs more abundantly when estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of estrogen-activating enzymes and/or deficient expression of deactivating (protective) enzymes. Excessive formation of E(1)(E(2))-3,4-Q is the result of this imbalance. Oxidation of catechols to semiquinones and quinones is a mechanism of tumor initiation not only for endogenous estrogens, but also for synthetic estrogens such as hexestrol and diethylstilbestrol, a human carcinogen. This mechanism is also involved in the initiation of leukemia by benzene, rat olfactory tumors by naphthalene, and neurodegenerative diseases such as Parkinson's disease by dopamine. In fact, dopamine quinone reacts with DNA similarly to the E(1)(E(2))-3,4-Q, forming analogous depurinating N3Ade and N7Gua adducts. The depurinating adducts that migrate from cells and can be found in body fluids can also serve as biomarkers of cancer risk. In fact, a higher level of estrogen-DNA adducts has been found in the urine of men with prostate cancer and in women with breast cancer compared to healthy controls. This unifying mechanism of the origin of cancer and other diseases suggests preventive strategies based on the level of depurinating DNA adducts that generate the first critical step in the initiation of diseases.
110 citations
TL;DR: It is shown that Polζ contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in responseto malfunction of normal replicative machinery due to mutations in replication genes.
Abstract: DNA polymerase ζ (Polζ) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polζ to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polζ-dependent damage-induced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polζ contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polζ-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polζ, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polζ separate from its modification at Lys164.
95 citations
TL;DR: It is shown that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K-Rac1-JNK signaling and inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I-induced N- caderin up-regulation.
Abstract: During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I–induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I–induced N-cadherin up-regulation. These data implicate PI3K–Rac1–JNK signaling in collagen I–induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I–induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I–induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K–Rac1–JNK signaling.
TL;DR: Treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A.T to G.C mutations in the DNA of the mammary gland of ACI rats, demonstrating estrogen genotoxicity is mutagenic in the female ACI rat, a model for estrogen-dependent breast cancer.
TL;DR: The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well, and indicates a broader application of formic acid than cyanogen bromide in cleaving fusion proteins.
TL;DR: It is shown that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization, and KSR1 is a member of a unique class of proteins whose deletion blocks bothsenescence and transformation.
Abstract: In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.
TL;DR: Phenylglycine with unique chemical shifts with potential future use for NMR studies may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides.
TL;DR: Results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs and show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking.
Abstract: Using a defined substrate DNA with a single psoralen interstrand cross-link (ICL), we studied the molecular mechanism of human ICL repair. In vitro ICL repair by human extracts is dependent on replication and is a largely error-free process. Extracts from a human BRCA2-defective mutant cell line, CAPAN-1, are severely compromised in ICL repair. Specifically, 'unhooked' but not fully repaired products accumulate in the reaction with CAPAN-1, and transient expression of BRCA2 in CAPAN-1 restores repair activity. Together, these results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs. We also show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking. This study provides new mechanistic insights into the repair of ICLs in human cells.
TL;DR: It is found that the induction of arthritis is impaired in the absence of KSR, and KSR plays a role in ERK activation during inflammatory and stress responses both in vitro and in vivo.
Abstract: The MAPK ERK is required for LPS-induced TNF production by macrophages. Although the scaffold kinase suppressor of Ras (KSR)1 is required for efficient Erk activation by mitogenic stimuli, the role of KSR1 in ERK activation by inflammatory and stress stimuli is unknown. In this study, we examined the effects of KSR deficiency on ERK activation by stress stimuli and show that ERK activation by TNF, IL-1, and sorbitol is attenuated in the absence of KSR1. To determine the significance of this defect in vivo, we tested KSR-deficient mice using a passive transfer model of arthritis. We found that the induction of arthritis is impaired in the absence of KSR. Thus, KSR plays a role in ERK activation during inflammatory and stress responses both in vitro and in vivo.
TL;DR: It is found that Aag(-/-) mice treated with a single high dose of STZ were protected from widespread beta-cell necrosis and diabetes, suggesting that N3-methyladenine adducts that accumulate in the genome may not be promutagenic in beta-cells.
TL;DR: Elongation complexes of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus are characterized and it is found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature and, depending upon the organization of the scaffold, possess distinct translocation conformations.
Abstract: We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50-80 degrees C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution.
TL;DR: It is suggested that CAG·CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.
Abstract: Cells of the central nervous system (CNS) are prone to the devastating consequences of trinucleotide repeat (TNR) expansion. Some CNS cells, including astrocytes, show substantial TNR instability in affected individuals. Since astrocyte enrichment occurs in brain regions sensitive to neurodegeneration and somatic TNR instability, immortalized SVG-A astrocytes were used as an ex vivo model to mimic TNR mutagenesis. Cultured astrocytes produced frequent (up to 2%) CAG.CTG contractions in a sequence-specific fashion, and an apparent threshold for instability was observed between 25 and 33 repeats. These results suggest that cultured astrocytes recapitulate key features of TNR mutagenesis. Furthermore, contractions were influenced by DNA replication through the repeat, suggesting that instability can arise by replication-based mechanisms in these cells. This is a crucial mechanistic point, since astrocytes in the CNS retain proliferative capacity throughout life and could be vulnerable to replication-mediated TNR instability. The presence of interruptions led to smaller but more frequent contractions, compared to a pure repeat, and the interruptions were sometimes deleted to form a perfect tract. In summary, we suggest that CAG.CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.
TL;DR: The mosaic structure of a single protein crystal was analyzed by reflection profiling and topography using highly parallel and monochromatic synchrotron radiation in this paper, where three reflections were collected with a digital topography system for three reflections in a region where the Lorentz effect was minimized.
Abstract: The mosaic structure of a single protein crystal was analyzed by reflection profiling and topography using highly parallel and monochromatic synchrotron radiation. Fine-φ-sliced diffraction images (0.002° stills) were collected using a conventional large-area CCD detector in order to calculate reflection profiles. Fine-φ-sliced topographic data (0.002°) stills were collected with a digital topography system for three reflections in a region where the Lorentz effect was minimized. At room temperature, several different mosaic domains were clearly visible within the crystal. Without altering the crystal orientation, the crystal was cryogenically frozen (cryocooled) and the experiment was repeated for the same three reflections. Topographs at cryogenic temperatures reveal a significantly increased mosaicity, while the original domain structure is maintained. A model for the observed changes during cryocooling is presented.
TL;DR: Compared with substrate-bound NTPase from a thermophilic organism, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group.
Abstract: The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound.
TL;DR: It is determined that the FGF‐4 promoter is highly responsive to a heterologous enhancer in both EC cell lines; whereas, the F GF‐4 enhancer is 7‐ to 10‐fold less active in P19 than in F9 EC cells, suggesting a mechanism used to decrease, but not abolish, FGF•4 expression as the early embryo develops.
Abstract: Transcription factors Oct-3/4 and Sox2 behave as global regulators during mammalian embryogenesis. They work together by binding co-operatively to closely spaced HMG and POU motifs (HMG/POU cassettes). Recently, it was suggested that a critical Sox2:Oct-3/4 target gene, FGF-4, is expressed at lower levels in P19 than in F9 embryonal carcinoma (EC) cells, due to lower levels of Sox2 in P19 than in F9 cells. We tested this possibility to better understand how FGF-4 expression is modulated during development. Although we found that P19 EC cells express ∼10-fold less FGF-4 mRNA than F9 EC cells, we determined that Sox2 levels do not differ markedly in F9 and P19 EC cells. We also determined that Sox2 and Oct-3/4 work together equally well in both EC cell lines. Moreover, in contrast to an earlier prediction based on in vitro binding studies, we demonstrate that the function of the HMG/POU cassettes of the FGF-4 and UTF1 genes does not differ significantly in these EC cell lines when tested in the context of a natural enhancer. Importantly, we determined that the FGF-4 promoter is highly responsive to a heterologous enhancer in both EC cell lines; whereas, the FGF-4 enhancer is 7- to 10-fold less active in P19 than in F9 EC cells. Because F9 and P19 EC cells are likely to represent cells at different stages of mammalian development, we suggest that this difference in FGF-4 enhancer activity may reflect a mechanism used to decrease, but not abolish, FGF-4 expression as the early embryo develops. J. Cell. Physiol. © 2006 Wiley-Liss, Inc.
TL;DR: The aim of this study was to determine the underlying genetic basis for epithelial-specific expression of ESX and to identify a 30bp ESX enhancer sequence (EES) approximately 3 kb upstream of the proximal promoter.
TL;DR: A Microsoft Windows-based system called Ripple has been developed to drive a digital topography camera and to collect and analyze a sequence of topographic images.
Abstract: A Microsoft Windows-based system called Ripple has been developed to drive a digital topography camera and to collect and analyze a sequence of topographic images. It has a graphical user interface, is easy to use and can be parasitically installed at existing beamlines. The modular design allows for the simple addition of detectors, detector image file formats, triggers and motion controllers.
TL;DR: The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3(1(2) and P6 (1(5)).
Abstract: The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P31(2)21 (a = b = 168.6, c = 149.8 A) and P61(5)22 (a = b = 130.9, c = 564.6 A). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.
15 Sep 2006
TL;DR: The sites of PAC reactions with DNA are mapped by treating PAC-damaged DNA with these enzymes to generate lesion-specific DNA strand breaks that are resolved by denaturing polyacrylamide gel electrophoresis.
Abstract: Polycyclic aromatic compounds (PACs) induce tumors by reacting with specific sequences of the initiating oncogene. The resulting DNA damage is converted into oncogenic mutation by in situ mutagenesis during repair or replication. The analysis of PAC/DNA reaction specificity is crucial for understanding the molecular biology of tumor initiation.
The sequence specificity of PAC/DNA reactions has been analyzed by various methods. These methods detect different types of DNA-damage lesions. For example, alkali-labile lesions can be mapped by a chemical procedure (piperidine treatment) and bulky stable adducts can be mapped by a photochemical method (laser-induced strand scission). Biochemical studies have identified several enzymes that act on damaged DNA and can be used for mapping the location of PAC/DNA reactions. For example, DNA lesions block the processive bypass of various enzymes (DNA polymerases, RNA polymerases and exonucleases), whereas some endonucleases (UvrABC exinuclease, S1 nuclease and apurinic/apyrimidinic (AP) endonuclease) make lesion-specific incisions. Therefore, the sites of PAC reactions with DNA are mapped by treating PAC-damaged DNA with these enzymes to generate lesion-specific DNA strand breaks that are resolved by denaturing polyacrylamide gel electrophoresis. More recently, various polymerase chain reactions (PCRs) have been developed for mapping the sequence preferences of PAC/DNA reactions in vivo. These techniques include ligation-mediated polymerase chain reactions (LMPCRs), single-strand ligation polymerase chain reactions (SSLPCRs) and terminal transferase-dependent polymerase chain reactions (TTDPCRs). In these methods, the PCR is used to amplify gene-specific DNA fragments generated by chemical or enzymatic DNA lesion processing.
Journal Article•
TL;DR: In this paper, a serial sacrifice experiment was conducted in which showed that the VPS treatment enhanced the development of large intestinal tumors induced by 1,2-DMH in animals sacrificed at 35 weeks after the first injection of the carcinogen.
Abstract: VPS, a hot water extract of the Coriolus versicolor mushroom, was given at a 2% dose level in the diet of female Swiss Webster CFW outbred mice in a serial sacrifice experiment. The mice were also administered either 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) as ten weekly subcutaneous (s.c) injections of 20 microg/g body weight or physiological saline (PS) as ten weekly (s.c) injections of 0.01 ml/g body weight. The animals were sacrificed at 26 weeks or 35 weeks after the first injection of 1,2-DMH or PS. The number of mice with large intestinal tumors and the total number of these tumors were: Group I (1,2-DMH), 29 and 438; Group 2 (VPS + 1,2-DMH), 29 and 344; Group 3 (VPS + PS), 0 and 0; and Group 4 (PS), I and 1, in the mice sacrificed at 26 weeks. The corresponding tumor incidences in mice sacrificed at 35 weeks were: Group 1 (1,2-DMH), 30 and 323; Group 2 (VPS + 1,2-DMH), 29 and 521; Group 3 (VPS + PS), 1 and 2; and Group 4 (PS), 0 and 0. Histopathologically, the tumors were diagnosed as polypoid adenomas and adenocarcinomas of the cecum, colon and rectum. Contrary to expectations, the VPS treatment enhanced the development of large intestinal tumors induced by 1,2-DMH in animals sacrificed at 35 weeks after the first injection of the carcinogen.
Journal Article•
TL;DR: Celecoxib treatment inhibited the development of large intestinal cancers in mice sacrificed at 26 or 35 weeks after the first injection of the carcinogen.
Abstract: In this serial sacrifice experiment, celecoxib (C) was administered at a 0.1% dose level, in the diet of female Swiss Webster CFW outbred mice. The animals also received either 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) as ten weekly subcutaneous (s.c.) injections at 20 Ig/g body weight or physiological saline (PS) as ten weekly s.c. injections at 0.01 ml/g body weight. Subsequently, the mice were sacrificed at 26 weeks or 35 weeks after the first injection of 1,2-DMH or PS. The number of mice with large intestinal tumors and the total number of these tumors were: Group 1 (1,2-DMH), 29 and 438; Group 2 (C + 1,2-DMH), 18 and 64; and Group 3 (PS), 1 and 1, in the mice sacrificed at 26 weeks. The corresponding tumor incidences in the mice sacrificed at 35 weeks were: Group 1 (1,2-DMH), 30 and 323; Group 2 (C + 1,2-DMH), 23 and 134; and Group 3 (PS), 0 and 0. Histopathologically, the tumors were diagnosed as polypoid adenomas and adenocarcinomas of the cecum, colon and rectum. Celecoxib treatment inhibited the development of large intestinal cancers in mice sacrificed at 26 or 35 weeks after the first injection of the carcinogen. In a recent study, we administered celecoxib (C), at a 0.1% dose level, in the diet of female Swiss Webster CFW outbred mice for life. The animals also received 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) as ten weekly subcutaneous (s.c.) injections at 20 Ig/g body weight. The administration of C reduced, in a statistically significant manner, the number of mice with large intestinal cancer and the total number of tumors (1). In the present series of experiments, essentially identical treatments were given to the same type of mice, except that the animals were sacrificed at either 26 or 35 weeks after the first injection of 1,2-DMH or physiological saline (PS). Serial sacrifice investigations are frequently employed to disclose the time sequence of cancer induction and progression in chemical carcinogenesis and prevention studies. Additionally, the current studies were part of some immunological experimentation in which the immune phenotype, cytokine expression and the reversal of tumor- associated immune suppression were altered. The induction of large intestinal tumors was associated with a significant increase in immature myeloid suppressor cells and a significant decrease in CD+T cells in the spleen, both of which were reversed by C administration (2). Thus, celecoxib, a non-steroidal anti-inflammatory agent selective for the cyclooxygenase-2 (COX-2) enzyme, which is essential to the synthesis of prostaglandins, was proven to prevent large intestinal carcinogenesis in experimental animals.