scholarly journals Prospects for p53-based cancer therapy.

2005 ◽  
Vol 52 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Tomasz Stokłosa ◽  
Jakub Gołab
Keyword(s):  
Human Tumor ◽  
P53 Gene ◽  
Great Promise ◽  
Mutant Form ◽  
Wild Type ◽  
P53 Pathway ◽  
Cycle Arrest ◽  
Stress Signals ◽  
Near Future

The p53 tumor suppressor plays the role of a cellular hub which gathers stress signals such as damage to DNA or hypoxia and translates them into a complex response. p53 exerts its action mainly as a potent transcription factor. The two major outcomes of p53 activity are highlighted: cell cycle arrest and apoptosis. During malignant transformation p53 or p53-pathway related molecules are disabled extremely often. Mutations in p53 gene are present in every second human tumor. A mutant form of p53 may not only negate the wild type p53 function but may play additional role in tumor progression. Therefore p53 represents a relatively unique and specific target for anticancer drug design. Current approaches include several different molecules able to restore p53 wild-type conformation and activity. Such small molecule drugs hold great promise in treating human tumors with dysfunction of p53 pathway in the near future.

1183Beta-Adrenoceptor activation increases cardiac galectin-3 levels via the hippo signaling pathway

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
X.-J Du ◽  
W B Zhao ◽  
Q Lu ◽  
M N Nguyen ◽  
M Ziemann ◽  
...  
Keyword(s):  
Hippo Pathway ◽  
Fold Increase ◽  
Signalling Pathway ◽  
Mutant Form ◽  
Wild Type ◽  
Galectin 3 ◽  
Β Blockers ◽  
The Hippo Pathway ◽  
Hippo Signalling

Abstract Background Galectin-3 (Gal-3) is a clinical biomarker for risk of cardiovascular disease and a disease mediator forming a therapeutic target. However, the mechanism(s) that regulate cardiac expression of Gal-3 remains unknown. Activation of the sympatho-β-adrenergic system is a hallmark of heart disease, but the relationship of βAR activation and cardiac content of Gal-3 remains unknown. Purpose To determine the role of βAR activation in regulating cardiac Gal-3 level and the responsible mechanism focusing on the Hippo signalling pathway. Methods Wild-type and Gal-3 gene deleted (Gal3-KO) mice were used. To test the role of the Hippo pathway, we used transgenic (TG) mouse strains with cardiac overexpression of mammalian-20-like sterile kinase 1 (Mst1, mammalian orthology of Drosophila Hippo kinase) either in wild-type form (TG-Mst1) or dominative-negative kinase dead mutant form (TG-dnMst1). Effects of β-antagonist (isoprenaline, ISO) and antagonists were determined. We measured phosphorylation (Ser127) of YAP as a transcription co-regulator acting as the main signal output of the Hippo pathway. Results In wild-type mice, treatment with ISO led to a time- and dose-dependent increase in cardiac expression of Gal-3 (Fig. A) accompanied by elevated circulating Gal-3 levels (Fig. B). ISO treatment stimulated cardiac expression of Mst1 and YAP hyper-phosphorylation (i.e. inactivation, Fig. C), indicating activation of the Hippo signalling. These effects of ISO were inhibited by β-blockers (propranolol, Prop; carvedilol, Carv; Fig. D,E). Relative to non-TG controls, ISO-induced expression of Gal-3 was inhibited by 75% in TG-dnMst1 mice (inactivated Mst1), but exaggerated by 7-fold in TG-Mst1 mice (activated Mst1). Mst1-TG mice had a 45-fold increase in Gal-3 content, YAP hyper-phosphorylation and enhanced pro-fibrotic signaling. In Mst1-TG mice, whilst blood Gal-3 level was unchanged, treatment with ISO (6 mg, 2 days) evoked a marked increase in cardiac and blood Gal-3 levels. Using rat cardiomyoblasts, we showed that ISO-mediated Mst1 expression and YAP phosphorylation were PKA-dependent and that siRNA-mediated YAP knockdown led to Gal-3 upregulation. The role of Gal-3 in mediating ISO-induced cardiomyopathy was examined by treating wild-type and Gal3-KO mice with ISO (30 mg/kg, 7 days). ISO-treated wild-type mice had 8-fold increase in cardiac Gal-3, ventricular dysfunction, fibrosis, hypertrophy and activated inflammatory or fibrotic signalling. All these changes, except hypertrophy, were abolished by Gal3-KO. beta-AR regulates galectin-3 Conclusion βAR stimulation increases cardiac expression of Gal-3 through activation of the Hippo signalling pathway. This is accompanied by elevated circulating Gal-3 level. βAR antagonists inhibited βAR-Mst1 (Hippo) signalling and cardiac Gal-3 expression, actions likely contributing to the overall efficacy of β-blockers. Acknowledgement/Funding NHMRC of Australia; Nature Science Fund of China

scholarly journals Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome.

1995 ◽  
Vol 15 (1) ◽  
pp. 425-432 ◽  
Author(s):  
J W Shay ◽  
G Tomlinson ◽  
M A Piatyszek ◽  
L S Gollahon
Keyword(s):  
Epithelial Cells ◽  
P53 Gene ◽  
Wild Type ◽  
Stromal Fibroblasts ◽  
Breast Epithelial Cells ◽  
Li Fraumeni Syndrome ◽  
Human Papillomavirus Type 16 ◽  
Breast Epithelial

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.

SIRT1 Inhibition Induces Apoptosis In Human CML Progenitors by Enhancing p53 Acetylation and Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 200-200
Author(s):  
Ling Li ◽  
Lisheng Wang ◽  
Tinisha McDonald ◽  
Aswani Bolla ◽  
Tessa L. Holyoake ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Transcriptional Activity ◽  
P53 Gene ◽  
K562 Cells ◽  
Wild Type ◽  
P53 Expression ◽  
Significant Growth ◽  
Cd34 Cells ◽  
Significant Growth Inhibition

Abstract Abstract 200 Imatinib mesylate (IM) treatment is effective in inhibiting CML primitive progenitor growth but induces only modest levels of apoptosis. Improved approaches to enhance elimination of residual CML progenitors in IM-treated patients are required. The NAD+ dependent deacetylase SIRT1 is a stress-response gene that is expressed at higher levels in CML compared to normal CD34+ progenitors. We have shown that inhibition of SIRT1 expression using lentivirus-mediated SIRT1 shRNA expression results in modest induction of apoptosis in CML progenitors and significantly enhanced apoptosis in combination with IM (Blood 2009, 114: 189). SIRT1 inhibition does not induce apoptosis in normal progenitors or increase their sensitivity to IM. SIRT1 can potentially regulate the acetylation of several transcription factors, including the p53 tumor suppressor protein. In contrast to several other cancers, p53 mutations are rare in CP CML, suggesting that p53 may still be subject to activation in CML progenitors. However we have observed that p53 levels are reduced in IM-treated CML CD34+ progenitors. We were therefore interested in investigating whether increased apoptosis of CML progenitors following SIRT1 inhibition was related to enhancement of p53 activity via protein acetylation. We observed that inhibition of SIRT1 using shRNA resulted in increased acetylation of p53 in CML CD34+ cells without increase in total p53 expression on both western blotting and flow cytometry. SIRT1 inhibition also increased p53 acetylation in IM-treated cells. Acetylated p53 was observed to localize to the nuclei of CML CD34+ cells on immunofluorescence microscopy. Q-PCR analysis revealed increased expression of the p53 transcriptional targets, GFI-1 and Necdin, in SIRT1 knockdown CML CD34+ cells (Necdin, Si versus Ctrl, 2.7±0.4 fold, p<0.05, n=3; GFI-1, Si versus Ctrl, 2.4±0.4 fold, p<0.05, n=3). These results suggest that SIRT1 inhibition results in increased p53 acetylation, nuclear localization and transcriptional activity in CML CD34+ cells. To further investigate the role of p53 in mediating the effects of SIRT1 inhibition we concomitantly knocked down both p53 and SIRT1 in CML CD34+ cells. Inhibition of p53 expression by lentivirus mediated delivery of p53 shRNA significantly enhanced growth and reduced apoptosis of SIRT1 knockdown CML CD34+ cells (14±2% apoptosis with SIRT1 knockdown, 7±2% apoptosis with combined SIRT1 and p53 knockdown, p<0.05, n=3). These results confirm an important role for p53 in SIRT1 mediated effects in CML progenitors. SIRT1 inhibition did not inhibit growth or induce apoptosis in CML blast crisis K562 cells, which are p53 null. To further determine the specific role of p53 acetylation in mediating SIRT1 effects, we expressed both wild type and acetylation-deficient p53 constructs in K562 cells. K562 cells ectopically expressing the wild type p53 gene demonstrated significant growth inhibition and apoptosis following SIRT1 knockdown (SIRT1 shRNA, 18±5% versus Ctrl shRNA, 8±3%, p<0.05), increased levels of acetylated p53, and enhanced transactivation of a p53 reporter containing the mdm2 promoter cloned upstream of the luciferase gene (p<0.05). In contrast, K562 cells transfected with an acetylation-defective p53 gene (with all eight acetylation sites mutated) did not demonstrate significant growth inhibition or apoptosis following SIRT1 inhibition. These results indicate that the inhibitory effect of SIRT1 on CML cells is dependent on p53 acetylation. We conclude that inhibition of SIRT1 enhances p53 acetylation and transcriptional activity resulting in enhanced apoptosis of CML progenitors. SIRT1 is a potentially druggable target, and several groups are actively developing SIRT1 inhibitory compounds. Activation of p53 via SIRT1 inhibition represents an attractive approach to eradicate CML stem cells in combination with IM or other treatments. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recent Advances in the Pathogenesis and Treatment of Chronic Lymphocytic Leukemia

PRILOZI ◽  
2014 ◽  
Vol 35 (3) ◽  
pp. 105-120
Author(s):  
Dimitar G. Efremov ◽  
Luca Laurenti
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Great Promise ◽  
Next Generation Sequencing Technology ◽  
Recent Advances ◽  
Leukaemic Cells ◽  
Near Future

AbstractChronic lymphocytic leukaemia (CLL) is a common lymphoid malignancy characterized by the expansion and progressive accumulation of mature autoreactive B lymphocytes. The disease is clinically heterogeneous and incurable by standard chemotherapy. A major feature of the disease is the marked dependence of the leukaemic cells on various microenvironmental stimuli, which promote leukaemia cell growth, survival, and drug-resistance. Recently, considerable progress has been made in the understanding of the molecular mechanisms that drive CLL. The identification of recurrent genetic lesions using next generation sequencing technology has provided new data on the pathophysiology of the disease and has improved its prognostication. The recognition of the critical role of the B cell receptor (BCR) in driving the disease has resulted in the development of BCR pathway inhibitors that have the potential to completely transform CLL treatment in the near future. Other novel therapeutic agents, such as BCL2 antagonists and chimeric antigen receptor (CAR)-modified T-cells, are also showing great promise in clinical trials. In this review, we summarize some of these recent advances, with a particular focus on the BCR and corresponding pathway inhibitors.

scholarly journals The role of FOXO3 in DNA damage response in thyrocytes

2011 ◽  
Vol 18 (5) ◽  
pp. 555-564 ◽  
Author(s):  
Antje Klagge ◽  
Carl Weidinger ◽  
Kerstin Krause ◽  
Beate Jessnitzer ◽  
Monika Gutknecht ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Cellular Response ◽  
Wild Type ◽  
Cycle Arrest ◽  
Damage Repair ◽  
Cell Clones ◽  
Human Wild Type

Members of the forkhead box-O (FOXO) transcription factors family play an important role in stress defence. FOXO3 deregulation has recently been identified as a hallmark of thyroid carcinogenesis. In this study, we explore the role of FOXO3 in defence of oxidative stress in normal thyrocytes. Stable rat thyroid cell lines were generated expressing either the human wild-type FOXO3, a constitutively activating FOXO3 mutant, or the empty control vector. Cell clones were characterised for proliferation, function and morphology. Hydrogen peroxide and UV irradiation were used to induce oxidative stress. Changes in FOXO3 activity, induction of cell cycle arrest or apoptosis and kinetics of DNA damage repair were analysed. Upregulation of FOXO3 in thyrocytes resulted in decreased proliferation and changes in morphology, but did not affect differentiation. Hydrogen peroxide stimulated the expression of the FOXO3 target genes growth arrest and DNA damage-inducible protein 45 α (Gadd45α) and Bcl-2 interacting mediator of cell death (BIM) and induced programmed cell death in cells with overexpression of the human wild-type FOXO3. In contrast, UV irradiation resulted in a distinct cellular response with activation of FOXO3-c-Jun-N-terminal kinase-Gadd45α signalling and induction of cell cycle arrest at the G2-M-checkpoint. This was accompanied by FOXO3-induced DNA damage repair as evidenced by lower DNA breaks over time in a comet assay in FOXO3 cell clones compared with control cells. In conclusion, FOXO3 is a pivotal relay in the coordination of the cellular response to genotoxic stress in the thyroid. Depending on the stimulus, FOXO3 induces either cell cycle arrest or apoptosis. Conversely, FOXO3 inactivation in thyroid cancers is consistent with genomic instability and loss of cell cycle control.

scholarly journals Vpr R36w and R77Q Mutations Alter HIV-1 Replication and Cytotoxicity in T Lymphocytes

2020 ◽  
Author(s):  
Antonio Solis-Leal ◽  
Dalton C. Karlinsey ◽  
J. Brandon Lopez ◽  
Vicente Planelles ◽  
Brian D. Poole ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Cycle Arrest ◽  
Wild Type ◽  
Helper T Cell ◽  
Cycle Arrest ◽  
Aids Progression ◽  
G2 Cell Cycle Arrest ◽  
Hiv 1

Abstract Background: Acquired immunodeficiency syndrome (AIDS) is caused when HIV depletes CD4+ helper T cell levels in infected patients. Distinct AIDS development rates have shown that there are Rapid Progressor (RP) and Long-Term Non-Progressor (LTNP) patients, but the circumstances governing these differences in the kinetics of helper T cell depletion are poorly understood. Mutations in the Viral Protein R (Vpr) gene have been suggested to have a direct impact on helper T cell depletion. Interactions of Vpr with both host and viral factors affect cellular activities such as cell cycle progression and apoptosis. The Vpr mutants R36W and R77Q have been associated with RP and LTNP phenotypes, respectively; however, these findings are still controversial. This study examines the effects that Vpr mutations have in the context of HIV-1 infection of the HUT78 T cell line, using replication-competent CXCR4-tropic virus strains. Results: Our results show a replication enhancement of the R36W mutant accompanied by increased cytotoxicity. Interestingly, the R77Q mutant showed a unique enhancement of apoptosis (measured by Annexin V and TUNEL staining) and G2 cell cycle arrest; these effects were not seen with WT, R36W or Vpr null viruses. Thus, point mutations in Vpr can exhibit profound differences in mechanisms and rates of cell killing. Conclusions: The vpr gene is thought to be an important virulence factor in Human Immunodeficiency Virus type 1 (HIV-1). Vpr polymorphisms have been associated with different rates of AIDS progression. However, there is controversy about the cytopathic and virulence phenotypes of Vpr mutants, with contradictory conclusions about the same mutants. Here, we examine the replication capacity, apoptotic induction, and G2 cell cycle arrest phenotypes of three vpr mutants compared to wild-type HIV-1. One mutant associated with rapid AIDS progression replicated more efficiently and killed cells more rapidly than wild-type HIV-1. Another mutant associated with slow AIDS progression triggered apoptosis more efficiently than wild-type HIV-1 and showed significant levels of G2 cell cycle arrest. These results shed additional light on the role of vpr polymorphisms in T cell killing by HIV-1 and may help to explain the role of Vpr in different rates of AIDS progression.

scholarly journals Loss of CNOT9 begets impairment in gastrulation leading to embryonic lethality

2020 ◽  
Author(s):  
Hemanta Sarmah ◽  
Kentaro Ito ◽  
Mari Kaneko ◽  
Takaya Abe ◽  
Tadashi Yamamoto
Keyword(s):  
Messenger Rna ◽  
Mrna Decay ◽  
Mutant Form ◽  
Wild Type ◽  
Expression Control ◽  
Gene Expression Control ◽  
Risc Complex ◽  
Extensive Cell Death ◽  
Extensive Cell

AbstractThe multi-subunit eukaryotic CCR4-NOT complex imparts gene expression control primarily via messenger RNA (mRNA) decay. Here, we present the role of subunit CNOT9 in target mRNA decay during embryonic development. CNOT9 null mice appear normal by the onset of gastrulation (E7.0), however, exhibit growth and differentiation defects accompanied by extensive cell death by embryonic day 9.5 (E9.5). Sox-2 Cre conditional CNOT9 knockout mice show almost identical phenotype with brief delay in onset and progression, suggesting defects to be epiblast-dominant. Among various identified targets, we show that Lefty2 mRNA expression is post-transcriptionally regulated by CNOT9. Lefty2 3’-UTR containing mRNA has significantly higher stability in cells expressing mutant form of CNOT9, relative to cells expressing wild-type CNOT9. In addition, CNOT9 primarily localizes within the cytoplasm and bridges interactions between the CCR4-NOT complex and miRNA-RISC complex in gastrulating embryos.

p53 functions as a cell cycle control protein in osteosarcomas

1990 ◽  
Vol 10 (11) ◽  
pp. 5772-5781
Author(s):  
L Diller ◽  
J Kassel ◽  
C E Nelson ◽  
M A Gryka ◽  
G Litwak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Control ◽  
P53 Gene ◽  
Tumor Formation ◽  
Wild Type ◽  
Gene Encoding ◽  
Wide Range ◽  
A Cell ◽  
Wild Type P53

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

scholarly journals Structures of p53 Cancer Mutants and Mechanism of Rescue by Second-site Suppressor Mutations

2005 ◽  
Vol 280 (16) ◽  
pp. 16030-16037 ◽  
Author(s):  
Andreas C. Joerger ◽  
Hwee Ching Ang ◽  
Dmitry B. Veprintsev ◽  
Caroline M. Blair ◽  
Alan R. Fersht
Keyword(s):  
Hot Spot ◽  
Human Tumor ◽  
Wild Type ◽  
Mutation Site ◽  
Structural Distortions ◽  
Suppressor Mutations ◽  
Oncogenic Mutations ◽  
Cancer Mutations ◽  
Intragenic Suppressor

We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the “structural” oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg168mimics the role of Arg249in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.

Genetic Absence of Thrombin Receptor Par4 Overcomes the Obligate Requirement of EPCR in Mouse Placenta

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 425-425
Author(s):  
Michelle M Storage ◽  
Jianzhong An ◽  
Helena Liang ◽  
Qiuhui Yang ◽  
Mark Zogg ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Fold Increase ◽  
Thrombin Receptor ◽  
Mutant Form ◽  
Wild Type ◽  
Placental Development ◽  
Transgenic Expression ◽  
Placental Failure

Abstract Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical role in placentation and the maintenance of pregnancy. Severe Protein C deficiency in the mother results in pregnancy failure in early gestation. Thrombomodulin (Thbd) or the Endothelial Protein C Receptor (EPCR/ProcR) gene deletions result in embryonic death, secondary to developmental and functional abnormalities of the placenta. These molecules play multiple roles in coagulation and inflammation. The mechanisms governing their role in placental development and maintenance of placental function remain to be fully understood. The objective of this work is to identify the critical functions of EPCR and Thbd that are required for placental development. Both Thbd and EPCR augment activated protein C generation, albeit to different extents. We have examined if reduced activated Protein C generation mediates placental abnormalities of EPCR- and Thbd-null mice. Activation of thrombin receptors expressed on platelets and trophoblast cells can also contribute to placental failure. We examined the role of thrombin receptor Par4 in placental failure of EPCR-null mice. Methods: To assess the role of a PC generation in placental phenotype of Thbd- and EPCR-null mice, we used a transgene to express a hyperactivatable form of murine protein C (hMPC) under the control of transthyretin promoter. Thrombin cleaves this mutant form of Protein C 30-fold more efficiently than wild type protein C, without requiring the cofactor function of thrombomodulin. Wild type mice expressing hMPC show 2-fold increase in PC and 3-fold increase in aPC levels. hMPC expression in PC-null mice restores their ability to carry pregnancies. Breeding strategies were used to generate hMPCtg ProcR+/- or hMPCtg Thbd+/- female mice. These were mated to ProcR+/- or Thbd+/- males, respectively, and survival of ProcR-/- and Thbd-/- embryos was analyzed. Similar genetic strategy was used to analyze the role of thrombin receptor Par4 in the demise of EPCR-null embryos. Placental phenotypes and embryonic survival was compared with experiments in which the mother was continuously infused with LMWH using a subcutaneous osmotic pump. Results: As previously reported, EPCR-null mice die before 10.5 days post coitum (dpc) (ProcR+/- intercrosses, out of 41 live embryos none were ProcR-/-, 10 were expected, 21 aborted not genotyped, 7 pregnancies analyzed at 11.5 dpc) and none are found at wean (out of 30 live pups none were ProcR-/-, 8 were expected, 5 litters analyzed). Transgenic expression of hMPC in the mother resulted in some live ProcR-/- embryos at 11.5 dpc (4 ProcR-/- out of 41 live embryos, 10 were expected, 15 aborted not genotyped, 7 pregnancies at 11.5 dpc) and pups at wean (2 ProcR-/- out of 28 live, 7 litters analyzed). Despite transgenic hMPC expression ProcR-/- embryos and pups were underrepresented (P=0.007, chi square GOF test). Surviving ProcR-/- embryos showed normal placental histology grossly comparable to littermate controls. Expression of hMPC in the mother did not ameliorate fetal death of Thbd-null mice (out of 38 live embryos none were Thbd-/-, 10 expected, 16 aborted not genotyped, 7 pregnancies at 9.5 dpc). Continuous infusion of LMWH also resulted in some live ProcR-/- embryos at 11.5 dpc (3 ProcR-/- out of 19 live embryos, 5 expected, 11 aborted not genotyped, 3 pregnancies analyzed), but two were growth retarded and all 3 placentae showed markedly reduced placental labyrinth formation. In contrast to transgenic expression of hMPC and treatment with LMWH, when Par4-/- ProcR+/- animals were intercrossed, ProcR-/- animals were born at an expected Mendelian frequency (7 ProcR-/- out of 35 live pups, 9 expected, 7 litters analyzed). Conclusions: Our results show that transgenic expression of hMPC allows normal placental development and rescues a fraction of EPCR-null embryos. Thus, placental defect of EPCR-null mice is in part mediated by reduced generation of aPC on placental cells. In contrast to the transgenic expression of hMPC and LMWH treatment, genetic absence of Par4 completely overcame the placental defect and allowed development of EPCR-null embryos. Further studies will clarify contributions of maternal versus fetal Par4 in this phenomenon. Disclosures No relevant conflicts of interest to declare.

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