Expression and protein localisation of IGF2 in the marsupial placenta

<p>Abstract</p> <p>Background</p> <p>In eutherian mammals, genomic imprinting is critical for normal placentation and embryo survival. <it>Insulin-like growth factor 2 </it>(<it>IGF2</it>) is imprinted in the placenta of both eutherians and marsupials, but its function, or that of any imprinted gene, has not been investigated in any marsupial. This study examines the role of <it>IGF2 </it>in the yolk sac placenta of the tammar wallaby, <it>Macropus eugenii</it>.</p> <p>Results</p> <p><it>IGF2 </it>mRNA and protein were produced in the marsupial placenta. Both IGF2 receptors were present in the placenta, and presumably mediate IGF2 mitogenic actions. <it>IGF2 </it>mRNA levels were highest in the vascular region of the yolk sac placenta. IGF2 increased <it>vascular endothelial growth factor </it>expression in placental explant cultures, suggesting that IGF2 promotes vascularisation of the yolk sac.</p> <p>Conclusion</p> <p>This is the first demonstration of a physiological role for any imprinted gene in marsupial placentation. The conserved imprinting of <it>IGF2</it> in this marsupial and in all eutherian species so far investigated, but not in monotremes, suggests that imprinting of this gene may have originated in the placenta of the therian ancestor.</p

Targeting the angiotensin II type 2 receptor (AT2R) in colorectal liver metastases

BACKGROUND: Blockade of the angiotensin (ANG) II type 1 receptor (AT1R) inhibits tumour growth in several cancers, including colorectal cancer (CRC) liver metastases. While AT1R blockade has been extensively studied, the potential of targeting the antagonistically acting AT2R in cancer has not been investigated. This study examined the effect of AT2R activation with the agonist CGP42112A in a mouse model of CRC liver metastases. RESULTS: In vitro, mouse CRC cell (MoCR) proliferation was inhibited by treatment with CGP42112A in a dose dependent manner while apoptosis was increased. Immunofluorescent staining for key signalling and secondary messengers, PLA2 and iNOS, were also increased by CGP42112A treatment in vitro. Immunohistochemical staining for proliferation (PCNA) and the apoptosis (active caspase 3) markers confirmed a CGP42112A-associated inhibition of proliferation and induction of apoptosis of mouse CRC cells (MoCR) in vivo. However, angiogenesis and vascular endothelial growth factor (VEGF) appeared to be increased by CGP42112A treatment in vivo. This increase in VEGF secretion by MoCRs was confirmed in vitro. Despite this apparent pro-angiogenic effect, a syngenic orthotopic mouse model of CRC liver metastases showed a reduction in liver to body weight ratio, an indication of tumour burden, following CGP42112A treatment compared to untreated controls. CONCLUSIONS: These results suggest that AT2R activation might provide a novel target to inhibit tumour growth. Its potential to stimulate angiogenesis could be compensated by combination with anti-angiogenic agents

Evolution of the CDKN1C-KCNQ1 imprinted domain

<p>Abstract</p> <p>Background</p> <p>Genomic imprinting occurs in both marsupial and eutherian mammals. The <it>CDKN1C </it>and <it>IGF2 </it>genes are both imprinted and syntenic in the mouse and human, but in marsupials only <it>IGF2 </it>is imprinted. This study examines the evolution of features that, in eutherians, regulate <it>CDKN1C </it>imprinting.</p> <p>Results</p> <p>Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that <it>CDKN1C </it>is syntenic with <it>IGF2</it>. However, there are fewer LTR and DNA elements in the region and in intron 9 of <it>KCNQ1</it>. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of <it>KCNQ1 </it>and promoter elements could not be detected, the antisense transcript <it>KCNQ1OT1 </it>that regulates <it>CDKN1C </it>imprinting in human and mouse is still expressed.</p> <p>Conclusion</p> <p>CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of <it>KCNQ1OT1 </it>in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that <it>CDKN1C </it>imprinting cannot be due to its synteny with <it>IGF2 </it>or with its placental expression in mammals.</p

Analysis of the platypus genome suggests a transposon origin for mammalian imprinting

Comparisons between the platypus and eutherian mammalian genomes provides new insights into how epigenetic imprinting may have evolved in mammalian genomes

Blockade of MMP14 Activity in Murine Breast Carcinomas: Implications for Macrophages, Vessels, and Radiotherapy

Background: Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. Methods: Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. Results: DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm3: control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. Conclusion: MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumor

IGF2 protein in the bilaminar (A and B) and trilaminar (D and E) yolk sac at day 25–26 of gestation

IgG negative controls for the bilaminar (C) and trilaminar (F) yolk sac. Staining was strongest in the trophoblast (Tr), but some endodermal (En) cells of the yolk sac placenta also stained. Staining was generally stronger in the trilaminar yolk sac and in both portions of the yolk sac staining increased later in gestation (see Fig. 2). Strong staining can be seen in the uterine epithelium (Ep) immediately adjacent to the bilaminar (avascular) yolk sac placenta. There was little staining in the mesenchyme (Me) and endothelium of large vitelline vessels (Vv) of the trilaminar (vascular) yolk sac placenta. Some stromal (St) and endothelial cells (Ed) in the maternal endometrium also stained (G and IgG negative H), as did the uterine epithelium (Ep) and some endometrial glands (Gl). Scale bar is shown at the bottom left of each image.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p

IGF2R (A & C) and IGF1R (E & G) protein in the bilaminar (BYS; A & E) and trilaminar (TYS; C & G) yolk sac at day 25

Appropriate IgG antibody negative controls for IGF2R and IGF1R antibodies are shown (B, D, F, & H). IGF2R staining was strongest in the trophoblast (Tr), with lighter staining in the yolk sac endoderm (En) and little or no staining in the mesenchyme (Me) surrounding vitelline vessels (Vv). IGF2R staining was localised in the cytoplasm and cell membrane. IGF1R stained all yolk sac cell types. Both antibodies also stained the uterine epithelium and some stromal cells in the endometrium (Endo). Scale bar is shown at the bottom left of each image.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p

Intensity of staining to IGF2 and IGF2R antibodies in the yolk sac trophoblast during the final third of gestation (A)

The intensity of staining was measured subjectively as described in the experimental procedures. The bilaminar (BYS) (shaded bars) and trilaminar yolk sac (TYS) (open bars) of matched samples were assessed independently. Samples were grouped into days 19–21 (n = 4), 22–24 (n = 5), and 25–26 (n = 4). Staining intensity to the IGF2 antibody was consistently stronger in the TYS, especially at days 25–26. Staining by the IGF2 antibody was notably lighter at days 19–21 than later stages (days 22 to 26). Staining by the IGF2R antibody did not differ notably between the bilaminar and trilaminar yolk sac, nor were there marked differences corresponding to developmental stage. Intensity of staining by IGF2 and IGF2R antibodies in yolk sac cells (B). Staining intensity was noticeably higher in the trophoblast (Tr) (stippled bars) than in the yolk sac endoderm (En) (striped bars) of the bilaminar (BYS) and trilaminar (TYS) for IGF2, but not IGF2R. The staining intensity represents the average for fetal stages between days 19 and 26 (n = 13). Light background staining with the IgG antibody negative control in the yolk sac endoderm was taken into account when judging the staining intensity of the yolk sac endoderm for IGF2 and IGF2R antibodies.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p