npg Cell Proliferation and Cell Cycle S36 Cell Research (2006) 16:S36-S53 npg © 2006 IBCB, SIBS, CAS All rights reserved 1001-0602/06 www.nature.com/cr APOCB2006-04-001 rate under different hypotonic solution in vitro. Methods: Inhibition of the mitotic kinesin Eg5 induces mitotic arrest Mechanical strain was applied by swelling osteoblat-like and apoptosis, and upregulates Hsp70 through the PI3K/Akt cells in varied hypotonic solutions, such as 300mOsm, pathway in multiple myeloma cells 277mOsm, 240mOsm and 163mOsm..Mitochondrial mem- Min Liu1, Ritu Aneja2, Harish C Joshi2, Jun Zhou1 brane potential(∆Ψm), cell apoptosis ratio, S stage percent, 1Department of Genetics and Cell Biology, Nankai University ALP and Ca2+ (concentration of endocellular calcium)were College of Life Sciences, Tianjin, China; 2Department of Cell used to assay pre-apoptosis, and cell proliferation in fol- Biology, Emory University School of Medicine, Atlanta, US lowing periods, such as 30 min, 2 h, 4 h, 6h, 12 h and 24 h. Results: Stretching MG63 osteoblast-like by swelling The microtubule-dependent motor protein Eg5 plays a in hypotonic solution could rapidly alter mitochondria critical role in spindle assembly and maintenance in mito- membrane potential, while the cell proliferation showed sis. Herein we show that suppression of Eg5 by a specific an distinct changes. But under the intense stretching , the inhibitor arrests mitosis, induces apoptosis, and upregulates mitochondrial membrane potential (∆Ψm) began to de- Hsp70 in human multiple myeloma cells. Mechanistically, press as well as the enhancement of apoptosis ratio. Laser Hsp70 induction occurs at the transcriptional level via a Confocal Microscope imaging of mitochondria stained cis-regulatory DNA element in Hsp70 promoter and is with JC-1, a trans-membrane potential-sensitive vital dye, mediated by the PI3K/Akt pathway. Eg5 inhibitor-mediated showed that ratio of red/green decreased under 163mOsm Hsp70 upregulation is cytoprotective, because blocking and 240 mOsm after exposed to stretching for 2 h and 4 h Hsp70 induction directly by antisense or small interfering respectively. The change of S stage percent was consistent RNA or indirectly by inhibiting the PI3K/Akt pathway with the changed ratio of red/green. With the prolonga- significantly increases Eg5 inhibitor-induced apoptosis. tion of stretching time, the changes of ALP and Ca2+ just Furthermore, a farnesyltransferase inhibitor interacts syn- showed different values in varied condition. Conclusion: ergistically with the Eg5 inhibitor in inducing apoptosis (1) The proper stretching may promote differentiation and through disrupting the Akt/Hsp70 signaling axis. These proliferation of osteoblast cell, and ALP shows an upward findings provide the first evidence for Eg5 inhibitor activity trend; (2) The overstress inhibits proliferation of osteoblasts in hematologic malignancy and identify Hsp70 upregula- and induces apoptosis of osteoblast-like; (3) The effect of tion as a critical mechanism responsible for modulating hypotonic solution on human osteoblast-like is similar to myeloma cell sensitivity to Eg5 inhibitors. In addition, that of the dynamic mechanical loading on bone cells re- these findings suggest that combination of Eg5 inhibitors ported; (4) The mitochondrial membrane potential (∆Ψm) with agents abrogating Hsp70 induction is more useful for may be an early sensitivity index of response to mechanical myeloma therapy in the clinic. strain, however, ALP and Ca2+ just only correspond to alter during the later period of stretching. Keywords: Eg5; mitosis; apoptosis; Hsp70; PI3K/Akt Correspondence: Jun Zhou Keywords: human osteoblast-like cell; hypotonic solution; E-mail: [email protected] mitochondrial membrane potential; proliferation; apoptosis Correspondence: Weiqun Zhang E-mail: [email protected] APOCB2006-04-002 Effect of hypotonic solution on the mitochondrial membrane potential and apoptosis in human ssteoblast-like MG63 APOCB2006-04-003 Weiqun Zhang1, Zhiming Zhu1, Yonglie Chao1, Yunqi Liu2, Murine mesenchymal stem cells isolated by low density Yu Lei1, Jun Cui1 primary culture system keep differentiation potential up to 1Department of Prosthodontics, West China College of Stomatol- passage 10 ogy , Sichuan University, Chengdu 610041, China; 2Department Mohamadreza B Eslaminejad1, Aghbibi Nikmahzar1, of Medicine, Binzhou Medical College, Binzhou, Shandong Leila Taghiyar2, Samad Nadri3, Mohamad Massumi1 256603, China 1Stem Cell Department, Royan Institute, Tehran, Iran; 2Devel- opmental Biology Department, Lorestan University, Khoram To investigate the involvement of mitochondrial mem- abad, Iran; 3Biochemistry Department, Payam Nour University, brane potential(∆Ψm) in the early response of human Tehran, Iran osteoblast-like MG63 to mechanical strain , and study the proliferation response to strain, ALP and apoptosis Murine mesenchymal stem cells (mMSCs) and the difficult Cell Research | www.cell-research.com npg S37 task of isolation and purification of them have been the APOCB2006-04-004 subject of rather extensive investigation. The present study Accumulation of p27kip1 is associated with RA-induced sought to isolate these cells from two different strains one growth arrest and neuronal differentiation of human neural outbred and the other inbred mouse, primarily through a progenitor vells relatively simple but novel approach, the most important Qiuyan Xu2, Yongmei Zhao2, Yisong Wang3, Haiyan Zhang1 feature of which was the low density primary culture of 1Department of Cell Biology, 3Department of Neurology Re- bone marrow cells. For this purpose, mononuclear cells pairing, the Beijing Center of Neural Regeneration and Beijing from either NMRI or Balb/c bone marrow were plated at Institute for Neuroscience, Capital Medical University, Beijing about 500 cells per well of 24-well plates and incubated for 100069, China; 2Beijing Research Laboratory for neural bio- 7 d. At this point, the fibroblastic clones that had emerged chemistry, Xuan-wu Hospital, Capital Medical University, Beijing were pooled together and expanded through several sub- 100053, China cultures. To investigate the mesenchymal nature, we dif- ferentiated the cells into the osteoblastic, chondrocytic and Neural transplantation is a promising strategy for of CNS adipocytic lineages in different subcultures up to passage injuries and neurodegenerative disorders. Human neural 10. In present investigation, the best culture condition for progenitor cells (hNPCs) are currently believed to have maximum proliferation and also the expression of certain important potential for clinical application. However,the surface marker on isolated cells were examined.Further- underlying molecular mechanism involved in cell cycle more; the colonogenic potential of the cells was tested by withdrawal and neuronal differentiation remains poorly Colony Forming Unit- Fibroblast Assay (CFU-F). Accord- defined. hNPCs can differentiate into neuronal phenotype ing to the results, one week after culture initiation, several following all-trans Retinoic acid (RA) treatment. In the clones each comprising several fibroblastic cells appeared present study, we demonstrate a functional link between in each plate. The cells from different passages were ca- RA and one of the Kip/Cip proteins p27Kip1 in the control pable of differentiating into corresponding skeletal tissues. of neuronal differentiation in hNPCs. Methods: The hNPCs The cell had maximum proliferation when being cultured were derived from embryonic ventral telencephlon and at density of 100 cell/cm2 in a DMEM medium containing cultured in serum-free medium supplied with EGF and 15% fetal calf serum (FCS).FACS analysis indicated that bFGF. When the cells are 80% confluent, hNPCs were more than 90% of the cells are CD44+. Sca-1 was expressed exposed to RA (1 μM) for 1, 3, 5, 7 days respectively. The in about 20% of the cells and C-kit, VCAM and CD34 properties of hNPCs were determined by using flow cy- were not detected. Almost 75 colon each consisting of 20- tometry analysis (FACS), immunocytochemistry, RT-PCR 70 cells was formed per each 100 cell from either strain and Western Blot. Results: Cell cycle analysis performed plated in 25 cm2-flasks. Two murine strains showed some by FACS showed that G0-G1 rate and apoptotic rate of differences in term of their growth and surface antigens. hNPCs increased significantly after exposure to RA for 3 Taken together, low density primary culture system seems d. The result of immunocytochemistry, RT-PCR and West- to be an appropriate and simple way for the isolation and ern Blot showed that the expression of p27Kip1 increased purification of fibroblastic cells from the heterogeneous significantly following RA treatment, with a peak at 5 mixture of bone marrow cells. The fact that the fibroblastic d, while the expression of p21Cip1 and skp2 decreased cells isolated through this approach were not only able to significantly following RA treatment. The expression of differentiate into osteoblasts, chondrocytes and adipocytes CDK2 and cyclin E didn't change significantly before and but also able to maintain this property even after being after RA treatment. Conclusion: Accumulation of p27kip1 subjected to several rounds of subcultures added weight to is associated with RA-induced growth arrest and neuronal the assumption that they were, indeed, the mesenchymal differentiation of human neural progenitor cells. Studies stem cells described elsewhere. have demonstrated that p27Kip1 plays a key