Analysis of mRNA expression during preimplantation development.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R M Schultz
Publications and source records attributed to R M Schultz.
Culture of preimplantation mouse embryos.
Explore the source record for details and available documents.
Animals↗
Reprogramming of gene expression during preimplantation development.
A fundamental problem in early mammalian development is the transformation of the highly differentiated oocyte into the totipotent blastomeres by the 2-cell stage. This remarkable transformation probably entails reprogramming the pattern of gene expression. The maternal-to-zygotic transition occurs during the 1-cell stage, i.e., 1-cell embryo is transcriptionally active. DNA replication is a likely locus of regulation, since DNA replication, with consequent nucleosome displacement, would serve to facilitate the access of maternally derived transcription factors to their cis-acting DNA-binding sequences. In fact, the first round of DNA replication is essential for the expression of two genes, eIF-1A and the transcription-requiring complex (TRC), as well as global transcription. A growing body of evidence suggests that a transcriptionally repressive state develops during the 2-cell stage. Interestingly, inhibiting the second round of DNA replication inhibits the decrease in expression of both the TRC and eIF-1A, as well as total endogenous gene expression. This repression may be linked to a change in chromatin structure, since treatment of 2-cell embryos with histone deacetylase inhibitors prevents the decrease in expression of the TRC and eIF-1A, and also inhibits the development of the transcriptionally repressive state for global gene expression. The findings that histone deacetylases can be targeted to chromatin provide a mechanism to link histone deacetylation with repression of gene expression. J. Exp. Zool. (Mol. Dev. Evol.) 285:276-282, 1999.
Animals↗
Novel cryptophycin antitumor agents: synthesis and cytotoxicity of fragment "B" analogues.
A general synthetic approach to novel cryptophycin analogues 6 is described. N-Hydroxysuccinimide active ester 15, a key common intermediate, was converted to beta-epoxide 6 in three steps, via initial coupling with unprotected amino acid 9, followed by deprotection/macrolactamization of acyclic precursor 16, and final oxidation of styrene 7 to install the C7-C8 beta-epoxide. Cryptophycin styrenes 7 and beta-epoxides 6, bearing diverse side chains in fragment "B", were evaluated for cytotoxic activity. beta-Epoxides 6, in general, were significantly more potent than the corresponding alpha-epoxides 17 and styrenes 7. A benzyl side chain was required for potent activity, with beta-epoxide 6u, possessing a 3-Cl,4-(dimethylamino)benzyl moiety, as the most potent cytotoxic agent prepared, with an IC(50) = 54 pM, only 2-fold less than that of Cryptophycin-52 (3).
Amides↗
Synthesis and biological evaluation of cryptophycin analogs with substitution at C-6 (fragment C region).
Analogs of the antitumor agents cryptophycins 1 and 8 with dialkyl substitution at C-6 (fragment C) were synthesized and evaluated for in vitro cytotoxicity against human leukemia cells (CCRF-CEM). The activity of these analogs decreased as the size of the substituents at C-6 increased. The C-6 spirocylopropyl compound (2g) was highly potent in vitro and showed excellent antitumor activity in animal models.
Animals↗
Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts.
Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.
3T3 Cells↗
Synthesis and biological evaluation of novel cryptophycin analogs with modification in the beta-alanine region.
Structure modification of the beta-alanine region (fragment C) of the potent antimitotic agent cryptophycin was investigated. This includes: (1) introduction of substituents at the previously unsubstituted C7 position of the macrolide ring and (2) replacement of the (2R)-3-amino-2-methyl-propanoic acid (beta-alanine) with various (1)-amino acids to give the corresponding 15-membered unnatural cryptophycin analogs.
Amino Acid Substitution↗
The synthesis and biological activity of a series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates as antineoplastic and antiarthritic agents.
A new series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates 1-3 were synthesized through an efficient conversion of 2-pivaloyl-4-oxo-6-ethynylpyrido[2,3-d]pyrimidine 5 to the corresponding 4-amino analog 7 via the activated 1,2,4-triazole intermediate 6. Compound 7 was used as the key intermediate for the preparation of the final products. The detailed biological evaluation of these compounds both as antineoplastic and antiarthritic agents will be discussed.
Animals↗
Assessment of DNA damage in individual hamster embryos by comet assay.
DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single-strand breaks in DNA, a significant increase in DNA damage was detected in individual one-cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one-cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO(2) in air at 37 degrees C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one- and two-cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro.
Animals↗
DNA replication in the 1-cell mouse embryo: stimulatory effect of histone acetylation.
Temporal and spatial distribution of the sites of DNA replication were examined in 1-cell mouse embryos. Embryos were labelled with bromodeoxyuridine (BrdU) at hourly intervals after fertilisation, and the incorporation of BrdU was examined by laser-scanning confocal microscopy following immunostaining with an anti-BrdU antibody. DNA replication first started uniformly in both the male and female pronuclei in the intranuclear region and then was observed in the peripheral regions of nucleus and nucleolus. These changes, however, occurred asynchronously in that the female pronucleus required a longer time to complete replication in the intranuclear region but not in the peripheral regions. Inhibiting transcription with alpha-amanitin had no effect on the temporal and spatial patterns of DNA replication. Treatment of the embryos with trapoxin, a specific inhibitor of histone deacetylase, accelerated the completion of replication in the peripheral regions but not in the intranuclear region. These results suggest that DNA replication is temporally and spatially regulated in the 1-cell embryos and that acetylation of histones, but not transcription, is involved in the regulation of DNA replication.
Acetylation↗
Cellular pharmacology of MTA: a correlation of MTA-induced cellular toxicity and in vitro enzyme inhibition with its effect on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.
The mechanism of action of an antifolate may be investigated using a variety of experimental methods. These include experiments in a cell culture setting to observe possible protection against drug effects afforded by the end products of metabolic pathways, assessing the activity of purified target enzymes in the presence of the antifolate, and, finally, the measurement of drug effects on intracellular folate and nucleoside triphosphate pools. The current discussion is focused on studies using CCRF-CEM leukemia cells that were designed to compare and contrast mechanisms of action of the antifolates methotrexate, which is primarily a dihydrofolate reductase inhibitor, raltitrexed, a thymidylate synthase inhibitor, LY309887, a glycinamide ribonucleotide formyltransferase inhibitor, and MTA (multitargeted antifolate), which is a novel antifolate antimetabolite. The results of these studies support the hypothesis that MTA affects multiple enzymatic targets and has a distinct mechanism of action from methotrexate, raltitrexed, and LY309887.
Acid Anhydride Hydrolases↗
MTA (LY231514) in combination treatment regimens using human tumor xenografts and the EMT-6 murine mammary carcinoma.
An important component in the development of a new anticancer drug is an understanding of its potential for inclusion in combination treatment regimens. LY231514, a multitargeted antifolate (MTA), was tested in combination with cisplatin, methotrexate, 5-fluorouracil, paclitaxel, docetaxel, doxorubicin, LY329201 (a glycinamide ribonucleotide formyltransferase [GARFT] inhibitor), and fractionated radiation therapy in vivo using EMT-6 mammary carcinoma, human HCT 116 colon carcinoma, and human H460 nonsmall cell lung carcinoma grown as xenografts in nude mice. Isobologram methodology was used to determine the additivity or synergy of the combination regimens. MTA administered with cisplatin, paclitaxel, docetaxel, or fractionated radiation therapy produced additive to greater than additive tumor response by tumor cell survival assay and tumor growth delay. While an additive tumor response was observed when MTA was administered with methotrexate, synergistic tumor responses were seen when MTA was administered with the GARFT inhibitor, LY329201, or with the topoisomerase I inhibitor, irinotecan. MTA was administered in combination with full doses of each anticancer agent studied, with no evidence of increased toxicity resulting from the combination.
Animals↗
Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs.
Prior studies have indicated that MTA requires intracellular polyglutamation for optimal cytotoxic effect and that these polyglutamates potently inhibit several key enzymes of folate metabolism, including thymidylate synthase (TS), dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). In the present studies, we have investigated the mechanistic basis for resistance to MTA in several human tumor cell lines. The cell lines were developed for resistance by the gradual exposure to stepwise (fivefold) increases in the concentration of MTA over a 5-month period. The degree of resistance was 140-fold for GC3 colon carcinoma, 117-fold for HCT-8 ileocecal carcinoma, and 729-fold for CCRF-CEM leukemia cells adapted to 2 micromol/L MTA. The lines had strong cross-resistance (>3,200-fold) to raltitrexed. Only modest resistance was noted for methotrexate and the GARFT inhibitor, LY309887. The cytotoxicity of MTA in wild-type cells was only partially alleviated by thymidine addition (5 micromol/L) and complete protection required the addition of both hypoxanthine (100 micromol/L) and thymidine. In contrast, thymidine alone totally lacked protective activity in the MTA-resistant lines. The cells either demonstrated a GARFT-like reversal pattern (complete protection by hypoxanthine) for GC3MTA or a dihydrofolate reductase-like reversal pattern (complete protection by the combination of hypoxanthine and thymidine) for HCT-8MTA and CCRF-CEM(MTA) cells. Cellular resistance was multifactorial and stable on removal of selective pressure. Only GC3MTA cells showed increased TS activity (approximately 40-fold). Accumulations of 3H-MTA at 24 hours in CCRF-CEM(MTA), HCT-8MTA, and GC3MTA cells were 2%, 6%, and 46% of wild-type values, respectively. We also evaluated the cytotoxic activity of MTA in MCF-7 breast carcinoma and H630 colon carcinoma cells selected for resistance to raltitrexed and 5-fluorouracil, respectively, via TS amplification (provided by Dr P.G. Johnston, Belfast, Ireland). These cells demonstrated more than 200-fold less resistance to MTA compared with raltitrexed and MTA-induced cytotoxicity was prevented by hypoxanthine. These studies suggest that in addition to TS modulation, secondary targets emerge during the development of MTA resistance.
Antimetabolites, Antineoplastic↗
Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies.
The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.
Animals↗
Molecular cloning and expression of the mouse translation initiation factor eIF-1A.
Prior to determining the molecular basis for the transient increase in expression of eIF-1A during the 2-cell stage of the pre-implantation mouse embryo, we determined the sequence of full-length cDNA and defined properties of the genomic organization of the mouse eIF-1A gene. Northern blot analysis distinguishes three transcripts in mouse liver of 2.8, 2.2 and 1.9 kb in size. The three transcripts arise from initiation at two putative promoters separated by 627 bp. Initiation from the putative distal promoter yields both the 2.8 and 1.9 kb transcripts, in which the 1.9 kb transcript is generated by alternative splicing of 840 bp of intervening RNA. The putative distal promoter, which lacks both a TATA box and CCAAT box control elements but contains several GC-rich clusters, initiates transcription at two start sites that are separated by 30 bp. Thus, four transcripts are generated from the distal promoter. The putative proximal promoter that directs transcription of a single 2.2 kb mRNA is preceded by a TATA box element that binds TBP. Each of the promoters is used by the pre-implantation mouse embryo, since we have been able to amplify selectively each of the five individual eIF-1A transcripts initiated from each promoter and start site in the 2-cell mouse embryo.
5' Untranslated Regions↗
Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs.
Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.
Actins↗
Evidence that Gq family G proteins do not function in mouse egg activation at fertilization.
Embryonic development is initiated after the fertilizing sperm contacts the egg and triggers a process termed "egg activation," resulting in calcium release, cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. Heterotrimeric guanine nucleotide-binding proteins (G proteins) may be involved in mouse egg activation since inhibition of G protein beta gamma subunits partially inhibits sperm-induced cell cycle resumption. In addition, specific events of egg activation can be initiated in the absence of sperm by acetylcholine stimulation of mouse eggs overexpressing the human m1 muscarinic receptor, a G protein-coupled receptor. In somatic cell, G proteins in the Gq family couple ligand stimulation of the m1 muscarinic receptor to activation of phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated release of intracellular calcium. Since IP3-mediated calcium release is involved in egg activation at fertilization, we have examined the role of Gq family G proteins in both sperm-independent (muscarinic receptor-mediated) and sperm-induced egg activation using a function-blocking antibody raised against the common C-terminal region of Gq and G11 proteins. We show that this antibody effectively inhibits Gq family G proteins in mouse eggs by demonstrating that the antibody inhibits egg activation in response to stimulation of the m1 muscarinic receptor. This same antibody, however, does not inhibit sperm-induced egg activation events. These results indicate that although activation of Gq family G proteins can result in egg activation in the mouse, it is unlikely that these proteins are used by the sperm to initiate egg activation at fertilization.
Animals↗
Role of deoxycytidine kinase (dCK), thymidine kinase 2 (TK2), and deoxycytidine deaminase (dCDA) in the antitumor activity of gemcitabine (dFdC).
Explore the source record for details and available documents.
Animals↗