Key Points Mutant CALR induces TPO-independent growth in the human megakaryocytic cell line UT-7/TPO. Mutant CALR binds to the TPO receptor, inducing phosphorylation of JAK2 and activating downstream signaling.

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Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.

The complex strain-optical ratio, O*(ω), and the complex Young's modulus, E*(ω), of four vinyl polymers, atactic polypropylene, 1,2-polybutadiene, poly(ethylethylene), and poly(4-vinylbiphenyl), were measured from the rubbery plateau to the glassy zone to investigate the origin of the birefringence and the chain orientation in the glassy zone. The data, including those for polystyrene and poly(vinyl naphthalene), were analyzed through a modified stress-optical rule:  The rule states that O*(ω) and E*(ω) are described as a sum of two component functions (denoted by R and G) and that a proportional rule similar to the ordinary stress-optical rule holds well for each of the R and G components with different stress-optical coefficients. The R and G components have been interpreted to be respectively related to the orientation of the main chain axis toward the stretch direction and tilting of the structural units by a rotational orientation about the main chain axis. According to this interpretation, the stress-optical coefficients for the R and G components are predicted to decrease and increase, respectively, with increasing size of the side chain. The present study shows the validity of this prediction. In addition, it is suggested that the degree of the rotational orientation responsible for the G component becomes larger with increasing size of the side chain.

Key words albumin; CYP1A1; human hepatoblastoma HepG2 cell; size control; spheroid Three-dimensional multicellular spheroids possess greater in vivo tissue-like morphology and function than two-dimensional monolayered cells. Therefore, multicellular spheroids are considered useful in vitro systems for drug screening, including tests of drug metabolism and toxicity, [1][2][3][4][5][6] and for studying tumor metastasis. 7,8) Furthermore, such spheroids have attracted much attention for application in cell-based therapy [9][10][11] and as a building block for the construction of large tissues. 12,13) In a previous study, we demonstrated that spheroid formation greatly prolonged the survival of insulinsecreting NIT-1 cells after transplantation in mice. 9)Spheroid size is a key parameter to determine the functions and properties of spheroids. It is reported that oxygen concentration is decreased in the core of the spheroids of human hepatoblastoma HepG2 cells, hepatocytes, and tumor cells. [14][15][16][17] This affects the expression of various molecules, including hypoxia inducible factor (HIF)-1. In addition, paracellular or transcellular transport is required in order for the inner cells to take up extracellular materials and to release secretory products into the culture media. Despite their importance, details regarding the relationship between spheroid size and cellular functions remain to be elucidated. In particular, few studies have investigated the effect of spheroid sizes over 300 µm on their functions and properties.Investigating the effects of spheroid size requires the preparation of size-controlled, multicellular spheroids. Several techniques for spheroid formation have been reported, including the spinner flask, hanging drop, and liquid overlay methods. [18][19][20] However, a common problem with these techniques is the difficulty in controlling spheroid size. In contrast, microfabrication is suitable for the development of sizecontrolled microwells with narrow size distribution. 10,21,22) In a previous study, we demonstrated that polydimethylsiloxane (PDMS)-based microwells constructed using a microfabrication technique could be used to obtain spheroids with a very narrow size distribution. 23) We also found that coating the microwells with poly(N-isopropylacrylamide) (PNIPAAm), a thermoresponsive polymer, increased the quality of the spheroids, since the coating prevented the cells from adhering to the PDMS-based microwells. 23)In the present study, we developed PDMS-based microwells of different sizes in order to prepare variably sized multicellular spheroids. HepG2 cells were used for the study as they represent one of the most extensively used cell lines to evaluate drug metabolism and toxicity. 3,24,25) Production of various proteins is an index of liver function. Albumin is the most abundant serum protein produced by hepatocytes. 26)Another important liver function is the metabolism of various compounds, including drugs. CYP enzymes are the major enzymes involved in drug metabolism. CYP1A1 is ...

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

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