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At least 181 records · Page 10Linked to original sources

PubMed2006

Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK).

Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.

Amides↗

PubMed1995

Amnio-allantoic fluid exchange for the prevention of intestinal damage in gastroschisis: an experimental study on chick embryos.

In gastroschisis, the protruding intestine is usually shortened, thickened, and covered with a fibrous peel. Urine components, which are increased in the amniotic fluid with the onset of renal function, are responsible for these changes. In the chick embryo model of gastroschisis, the authors performed amnio-allantoic fluid exchange to reduce the effect of urinary products. There were three study groups: control, gastroschisis only, and gastroschisis plus exchange. There was a significant decrease in creatinine (P < .05) and a slight decrease in urea concentrations in the gastroschisis-plus-exchange group compared with the gastroschisis-only group. Whereas macroscopic and microscopic studies showed severe fibrosis and bowel-wall thickening in the latter group, the bowel was normal or slightly oedematous in the former. On the basis of this study, the authors suggest that antenatal amniotic fluid exchange might reduce the bowel damage in human foetuses.

Abdominal Muscles↗

PubMed2000

Subcellular localization of presenilins during mouse preimplantation development.

The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome-kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.

Acetylcysteine↗

PubMed1999

The role of plasminogen activator receptor in cancer invasion and dormancy.

Urokinase plasminogen activator receptor (uPAR) has been identified some 15 years ago and the anticipation was that its presence on the cell surface will provide a focus for anchoring uPA and possibly protect the enzyme from native inhibitors. The studies of the last decade have shown that uPA localized to the surface of cells by uPAR is indeed an important factor in the process of cancer cell invasion and metastasis. We developed a chick embryo model in which we showed that uPAR is crucial in invasion of stroma and in intravasation (breaching of the blood vessels walls). More recently and unexpectedly, uPAR--a protein anchored in the outer leaf-let of the plasma membrane, has been shown to initiate signal transduction events and affect cell migration. We have shown that uPAR co-associates with fibronectin binding integrin, alpha 5 beta 1, activates them and that this interaction leads to a greatly increased level of active ERK. When the association between uPAR and integrin or integrin and fibronectin are interrupted either by reduction of surface uPAR expression, or by other means, human carcinoma cells enter a state of protracted dormancy. We show that very high levels of active ERK are required to keep cancer cells proliferating in vivo.

Animals↗

PubMed1992

Role of neovasculature and vascular permeability on the tumor retention of photodynamic agents.

A variety of photodynamic sensitizers (chloroaluminum sulfonated phthalocyanine, tetraphenyl porphine sulfonate, mono-L-aspartyl chlorin e6, Photofrin, chlorin e6, and Uroporphyrin dihydrochloride I) were characterized by their ability to be retained in EMT-6 tumors growing in BALB/c mice. Two properties uniquely associated with tumors, proliferating neovasculature and vascular permeability, were tested for their relative importance in retaining the photosensitizer. A chick embryo model was used to compare photosensitizer uptake/retention in proliferating and nonproliferating neovasculature with retention in proliferating nonvascular tissue. Our results provide evidence that photosensitizers which are preferentially retained by tumors have a selective affinity for proliferating neovasculature. The chloroaluminum sulfonated phthalocyanine and tetraphenyl porphine sulfonate compounds possess the greatest affinity for proliferating neovasculature relative to nonvascular tissue, while the phthalocyanine has the largest tumor/normal differential in vivo of all the photosensitizers tested. Chlorin e6 and uroporphyrin dihydrochloride I were the only photosensitizers which were not retained in greater amounts by tumor tissues relative to normal tissues. Using a delayed-type hypersensitivity reaction, extended and constant vascular permeability was induced in BALB/c mice. Vascular permeability was quantitated by Evans blue extraction from the delayed-type hypersensitivity sites. Interestingly, leaky vessels alone did not result in photosensitizer retention, as seen with tumors. These data demonstrate that tumor-retained photosensitizers possess a selective affinity for proliferating neovasculature and that vascular permeability alone is not sufficient to retain these sensitizers.

Allantois↗

PubMed1996

Selective mitomycin C and cyclophosphamide induction of apoptosis in differentiating B lymphocytes compared to T lymphocytes in vivo.

Differentiating B and T lymphocytes differ in sensitivity to a number of environmental toxins and anticancer agents. B lymphocytes are susceptible and T lymphocytes resistant to killing by cyclophosphamide (Cy) metabolites capable of forming DNA interstrand cross-links. However, the mechanisms responsible for the rapid killing and loss of bursal-resident B lymphocytes are unknown. Therefore, we investigated the cellular mechanisms of selective toxicity of two cross-linking drugs, mitomycin C (MMC) and Cy, towards differentiating B and T lymphocyte populations using the chicken embryo model system. Viability of bursal-resident B lymphocytes (bursacytes) decreased starting at 5 h post exposure (PE) to MMC, and was maximally reduced by 71.6% by 10 h PE at the highest dose examined (9.0 micrograms MMC/g). Dose-dependent increases in the percentage of apoptotic bursacytes were observed as early as 5 h PE, and increased to 72% by 10 h PE. This was accompanied by reductions in bursacyte numbers. Cy also induced apoptosis in bursacytes. In contrast, thymus-resident lymphocytes (thymocytes) were much more resistant to the toxic effects of MMC and Cy. Viability of thymocytes was reduced by only 10% in the 9.0 micrograms/g MMC treatment group. In addition, the percentage of thymocytes engaged in apoptosis was much lower than that for bursacytes. MMC induced modest cell cycle inhibition in bursacytes and thymocytes. These data strongly suggest that MMC and Cy-induced diferential toxicity involves primarily early and extensive triggering of apoptosis in differentiating B lymphocytes, leading to rapid reduction of lymphocyte numbers in the embryonic bursa.

Animals↗

PubMed2006

Induction of CYP1A and cyp2-mediated arachidonic acid epoxygenation and suppression of 20-hydroxyeicosatetraenoic acid by imidazole derivatives including the aromatase inhibitor vorozole.

Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

Animals↗

PubMed2003

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host.

Animals↗

PubMed1998

Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct.

The ability of both acute and chronic ethanol exposures to elicit cell death within specific embryonic and adult tissues is believed to partly underlie ethanol's pathogenicity; however, the mechanism underlying this cell death is unknown. This study partially characterized the mechanism of ethanol-induced neural crest cell death in a chick embryo model of fetal alcohol syndrome. In situ DNA end-labeling demonstrated this cell death was apoptotic and occurred at embryonic ethanol levels as low as 42 mM. Regardless of the initial exposure time, this apoptosis always appeared at a distinct developmental time point simultaneous with the normal deletion of a cranial neural crest subset. This suggested that ethanol might act through aberrant activation of the endogenous death pathway; however, ethanol exposure failed to induce two components of this pathway, the homeotic transcription factor msx-2 and the growth factor bone morphogenetic protein 4. Both endogenous and ethanol-induced death were blocked by local application of an interleukin-1beta converting enzyme/CED-3 protease (caspase) inhibitor, showing that the two paths converge mechanistically and suggesting the potential to prevent this aspect of ethanol's teratogenicity. Ethanol exposure did not significantly alter cell proliferation within neural crest-populated regions, suggesting that susceptibility to ethanol-induced death did not involve exit from the cell cycle. Apoptotic deletion of cranial neural crest could partially explain the craniofacial deficits characteristic of the fetal alcohol syndrome.

Adult↗

PubMed1997

Structure and function of embryonic growth plate in the absence of functioning skeletal muscle.

Normal growth and development of the skeleton require the presence of viable, actively contracting skeletal muscle throughout the fetal period. A chick embryo model of midgestation chemical paralysis and secondary muscle atrophy was used to test the hypothesis that functioning muscle stimulates the growth of long bones by influencing the proliferation, differentiation, and hypertrophy of chondrocytes in cartilage of the epiphysis and growth plate. Paralysis did not alter the overall developmental stage of the long bone or the organization of the growth plate. Compared with controls, however, uptake of bromodeoxyuridine in the paralyzed chick was reduced by 27-55% in the chondroepiphysis and uppermost zone of the tibial growth plate, indicating reduced proliferation of chondrocytes. A specific reduction in the size of the proliferative zone and a reduced number of proliferating cells were also observed. By contrast, in the second, post-proliferative zone of the growth plate, the height of the zone was unchanged and its area was only slightly reduced compared with controls. Finally, median hypertrophic cell profile area, a measure of cell size, was not significantly affected by paralysis, although frequency analysis revealed modest numerical reductions in the population of the largest hypertrophic chondrocytes in the paralyzed group. These data suggest that the role of functioning fetal muscle in maintaining proper skeletal growth may be mediated primarily through specific stimulation of the recruitment or proliferation of immature chondrocytes, or of both.

Animals↗

PubMed2005

Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.

The human tumor/chick embryo model involving grafting of human HT-1080 fibrosarcoma cells on the chorioallantoic membrane was used in conjunction with quantitative real-time Alu PCR to select in vivo a pair of isogenic cell lines (HT-hi/diss and HT-lo/diss), dramatically differing in their ability to disseminate from the primary tumor (i.e., intravasate into the chorioallantoic membrane vasculature and metastasize to the lungs). During an immunohistochemical time course study, HT-hi/diss cells were sequentially visualized having escaped from the primary tumors, engaged with the blood vessels, and eventually observed inside the chorioallantoic membrane capillaries, thus reflecting early intravasating events. In contrast, HT-lo/diss cells seemed restricted to their primary tumor. Importantly, after i.v. inoculation, both variants arrested, extravasated, and proliferated in host tissues with similar efficiencies, highlighting that the observed earlier events at the periphery of the primary tumor could account for their differential dissemination. In a mechanistic probing of these events, we determined that HT-hi/diss intravasation was sensitive to a broad-range matrix metalloproteinase (MMP) inhibitor. To analyze the possible role of individual MMPs, membrane-bound MMP-14 and secreted MMP-9 were individually down-regulated in HT-hi/diss cells with their corresponding small interfering RNAs. Despite efficient down-regulation of MMP-14, neither intravasation nor metastasis of HT-hi/diss cells was affected significantly. However, a substantial down-regulation of MMP-9 was accompanied by a surprising 3-fold increase in intravasation and metastasis. The results emphasize a rising awareness that targeting certain MMPs might result in an enhanced malignancy, exemplified herein at the intravasation level as this step of the metastatic cascade is dissected and quantified.

Animals↗

PubMed1993

Autosomal dominant tetramelic postaxial oligodactyly.

Postaxial limb deficiencies are most frequently unilateral and sporadic with deficiencies of the ulnar ray about one-third as common as those affecting the radial ray. Postaxial deficiency occurs in a number of genetic and sporadic syndromes, but isolated inherited tetramelic, postaxial oligodactyly has to our knowledge not been described. We report an affected mother and her 3 affected children from a 4-generation family with apparent autosomal dominant, non-syndromic, tetramelic, postaxial oligodactyly. The affected individuals manifest a very uniform pattern of postaxial deficiency ranging from complete absence of the 5th metacarpals, metatarsals, and phalanges to complete absence of the 5th metacarpals and metatarsals, but with some residual distal 5th phalanges. Limb buds may be considered a single developmental field with development proceeding rostral to caudal. Digit formation is apparently controlled by the zone of polarizing activity which releases a diffusible morphogen specifying positional information along the anterior/posterior axis. Recent investigations using chick and mouse embryo models suggest that 5' members of the Hox-4 gene cluster are responsible for interpreting this positional information to effect digit formation. The present family may represent a defect in Hox-4 gene patterning of hand and foot formation, an abnormality in morphogen gradient formation or alternately an inability of the most ulnar rays to respond to morphogen (receptor defect).

Child↗

PubMed1986

Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds.

The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay.

2-Acetylaminofluorene↗

PubMed1988

Effects of charcoal-extracted serum as a growth medium supplement on in vitro development of mouse embryos.

Two-cell mouse embryos (N = 952) were cultured in modified Ham's F-10 medium supplemented with 15% charcoal-extracted serum or 15% nonextracted serum from 17 patients. Each sample was assayed independently and all experiments run in duplicate. Ten nonextracted samples inhibited development to the blastocyst stage compared to controls in F-10 alone. Charcoal extraction significantly improved (P less than 0.05) development compared to nonextracted serum in eight of these samples. Seven samples supported development and no difference was noted between F-10 alone and extracted and nonextracted serum-supplemented media in this group. In conclusion, charcoal extraction significantly reversed the "embryotoxic" effects of some sera in the two-cell mouse embryo model.

Animals↗

PubMed1989

Ovarian hyperstimulation inhibits embryo implantation in the mouse.

Embryo implantation is dependent on the synchronous development of the embryo and of the endometrium. Pharmacologic doses of estrogens change endometrial histology and are known to inhibit implantation. During controlled ovarian hyperstimulation, such as occurs during an in vitro fertilization cycle, serum estradiol levels may be elevated to as much as three to six times those found during spontaneous cycles. Serum progesterone levels are also increased and may counteract the elevated estradiol levels. The overall effect of ovarian stimulation on implantation is therefore not known. To study this question, we developed a mouse embryo donation model. Donor embryos were obtained in the late morula to early blastocyst stage from hyperstimulated mated mice. The donated embryos were then transferred to the uteri of two groups of recipient mice. The study group underwent ovarian hyperstimulation with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) (OHR group), while the controls were allowed to cycle spontaneously (SR group). All recipient mice underwent cervical stimulation to induce a pseudopregnant state. Five embryos were transferred to the left uterine horn of each of nine OHR mice and seven SR mice. A higher implantation rate was noted in the SR group than in the OHR group (50 +/- 12 vs 8 +/- 4%, P less than 0.001). Our data suggest that, in the mouse, ovarian hyperstimulation impedes implantation by causing adverse changes in uterine receptivity.

Animals↗

PubMed2026

From stem cells to somites: Revealing genetic and exogenous factors of human embryogenesis.

Stem-cell-based human embryo models offer an ethically tractable platform for studying early human development. This study employs somitoids, three-dimensional models of human somitogenesis, to investigate how transcriptional programs and culture conditions influence somite formation and segmentation. We show that pre-differentiation culture medium impacts the developmental potential of induced pluripotent stem cells (iPSCs), with StemFit medium and Matrigel embedding outperforming mTeSR Plus medium in generating robust somite-like structures. Strikingly, these differences arise despite only subtle changes in transcriptomic and time-resolved proteomic profiles. P300-based proximity labeling also reveals a largely overlapping set of chromatin-associated regulators across iPSC conditions. In somitoids, enhancer-associated profiling highlights factors linked to somitogenesis, including MESP2 and TBX6. Knockout of three identified regulators, BPTF, RBPJ, and CITED2, demonstrate their essential roles in somite formation. Together, these findings highlight how culture conditions and enhancer-associated networks influence early human development and demonstrate somitoids as a scalable system for functional genomics.

Humans↗

PubMed2002

Apoptosis in the cortex of the developing mouse kidney.

Published levels of apoptosis in developing rat kidney (approximately 2.5%) seem large for a tissue with no obvious need for continual cell death. This paper examines the levels and patterns of apoptosis and mitosis in the cortical region of the developing metanephros of the mouse, the standard mammalian model embryo. Using confocal microscopy on specimens stained with propidium iodide to highlight nuclear morphology, optical sections of wholemount kidneys to a depth of approximately 50 microm were analysed and mitotic, apoptotic and interphase nuclei counted in the various compartments. Of the approximately 200 000 cells examined over E11.5-16.5, 2-3% were mitotic, confirming observations based on cryosections; the mitotic index peaked at E14.5, dropping to approximately 0.5% by P14. The mean apoptotic index during this period was 0.28%; this figure from wholemounts was approximately 10% of that earlier reported in cryosectioned rat kidneys. One possible explanation for the difference is that cryosectioning turns out to create small nuclear fragments that can stain strongly with propidium. Such fragments are not seen in wholemounts and do not stain with TUNEL. Wholemount mouse E11.5 tails and E16.5 lungs were also analysed and both their mitotic and their apoptotic indexes were similar to those in wholemount developing kidneys. These results show that the level of apoptosis in wholemount embryonic mouse kidney cortex is far less than previously reported in cryosectioned rat embryonic kidneys, and typical of that in other mouse embryonic tissues whose development seems not to require apoptosis.

Animals↗

PubMed1995

Multiplex polymerase chain reaction for sex determination of single mouse blastomeres.

Using a multiplex nested polymerase chain reaction (PCR) method with single copy genes and a dinucleotide repeat locus for the mouse Y and X chromosomes respectively, it was possible to discriminate between single cells derived from male and female embryos. Using single cells, amplification of Sry and Zfy sequences was not evident in all cases. It could be calculated that, with the PCR method used, 0.04% [95% (confidence interval 0.00-2.03)] of the male embryos would erroneously be diagnosed as female if analysis is performed on two cells. The calculated chance for total amplification failure, if two cells are used for analysis, would be 1.4% [95% (confidence interval 0.04-8.04)]. The mouse embryo model proved to be a helpful tool to develop skills in the application of PCR for preimplantation genetic diagnosis at the single cell level.

Animals↗