Survivin is a recently described inhibitor of apoptosis. Because suppression of apoptosis is important for carcinogenesis and tumor growth, we investigated the expression and function of survivin in human hepatocellular carcinomas (HCCs). We have shown that 4 HCC cell lines and 7 out of 8 human HCC tissues expressed survivin messenger RNA (mRNA), whereas expression of survivin mRNA was not detected in normal liver and nontumor areas of these tissues using the reverse transcription polymerase chain reaction. Survivin was detected primarily in the nucleus by immunofluorescence staining of HCC cells. In addition, 14 of 20 (70%) HCC tissues showed positive nuclear staining for survivin, whereas nontumor tissues showed little detectable staining by immunohistochemistry. Survivin expression strongly correlated with the proliferation index but not significantly with the apoptosis index in HCC tissues. Therefore, we performed cell cycle analysis after survivin transfection and showed that overexpression of survivin resulted in a decrease in the G 0 /G 1 phase and an increase in the S phase in all 4 HCC cell lines. Furthermore, we have found that survivin interacted with cyclin-dependent kinase 4 (Cdk4) and overexpression of survivin released p21 WAF1/Cip1 (p21) from Cdk4. From these results, we conclude that survivin promotes cell proliferation by interacting with Cdk4 and releasing p21 from Cdk4. This may play an important role in carcinogenesis and progression of human HCCs. (HEPA-TOLOGY 2000;31:1080-1085.)Regulation of programmed cell death is important in the preservation of homeostasis and morphogenesis of human tissues. 1,2 Impairment of apoptosis facilitates the accumulation of gene mutations by prolonging the cell cycle span and promoting resistance to immune-based cytotoxicity, 3 finally contributing to carcinogenesis. 4 It has been shown recently that the inhibitors of apoptosis proteins (IAP) are crucial regulators in the molecular mechanism of apoptosis. [5][6][7] Among the IAP members, survivin is unique in that it is undetectable in normal adult tissue, but abundantly expressed in transformed cells and a variety of human cancers. 8 It has also been shown that survivin inhibits apoptosis in cells exposed to diverse apoptotic stimuli 8-11 by associating with microtubules of the mitotic spindle 9 and inhibiting caspase activity. 10 Expression of survivin is highly correlated with prognosis in patients with neuroblastoma, 7,12 gastric cancer, 13 colorectal cancer, 14 and bladder cancers. 15 However, the biological functions of survivin, other than its antiapoptotic effect, are not well understood in human cancers.Therefore, to gain insight into additional roles of survivin in malignant cells, we investigated the expression and function of survivin in hepatocellular carcinoma (HCC) cell lines and human HCC. In this study, we showed that survivin expression correlates with HCC cell proliferation in the majority of human HCC tissues. Survivin overexpression obliterates the G 1 checkpoint by releasing p21 WAF1/Cip...

Caspase 3 is an essential death factor for the Fasmediated cell death, and its inactivation in cells is initiated by an interaction with p21 on mitochondria or with IAP family member ILP. Survivin is also a member of IAP family and is speci®cally expressed during embryogenesis and in tumor cells and suppresses cell death signaling. In our current study, we demonstrated that Survivin translocation into the nucleus is dependent on Fas stimulation and cell proliferation. Survivin also interacts with the cell cycle regulator Cdk4, leading to Cdk2/Cyclin E activation and Rb phosphorylation. As a result of Survivin/Cdk4 complex formation, p21 is released from its complex with Cdk4 and interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death. Here, we propose that Survivin supports procaspase 3/p21 complex formation as a result of interaction with Cdk4 resulting in suppression of cell death signaling.

Survivin is observed uniquely in tumor cells and developmental cells, which undergo either inappropriate or programmed cell growth. In the current study, we investigated the in¯uence of Survivin on cell cycle. Overexpression of Survivin resulted in accelerated S phase shift, resistance to G1 arrest, and activated Cdk2/ Cyclin E complex leading Rb phosphorylation. In addition, nuclear translocation of Survivin followed by an interaction with Cdk4 was detected. Interestingly, Survivin nuclear translocation coincided with S phase shift, and prevention of nuclear transport suppressed Survivin nuclear translocation and S phase shift. Further, we also observed that Survivin competitively interacted with the Cdk4/p16 INK4a complex in a cell free system and in vivo. These results suggest that Survivin initiates the cell cycle entry as a result of nuclear translocation followed by an interaction with Cdk4. Oncogene (2000) 19, 3225 ± 3234.

Caspase 3 is an essential factor for Fas-mediated cell death and exists endogenously in cells where its activation is suppressed by p21 and ILP. Inside the cell, procaspase 3 interacts with p21 on mitochondria. In the present study, we investigated the molecular basis for procaspase 3/p21 complex formation. During Fas-mediated cell death, mitochondria are damaged, accompanied by decreased mitochondrial membrane-potential and decreased intracellular ATP levels. This mitochondrial damage occurs before an estrangement of the procaspase 3/p21 complex, and we demonstrate that intracellular ATP-deprivation also initiates an estrangement of procaspase 3/p21 complex formation and accelerates Fas-mediated cell death. In addition, our current results revealed that the phosphorylated p21 by PKA interacts with procaspase 3. Here, we report that the mitochondrial role, especially for ATP synthesis, and PKA are necessary for the procaspase 3/p21 complex formation to resist Fas-mediated cell death. Cell Death and Differentiation (2000) 7, 721 ± 728.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-    1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N'-bis[N″, N‴-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.

Cell death induction by cytotoxic T lymphocytes (CTLs) is an important thesis for the understanding of tumor immunotherapy. In the current study we investigated the molecular machinery of CTL-induced cell death in human hepatocellular carcinoma cell lines (HCC lines). CTLs prepared from human peripheral blood induced cell death in all tested HCC lines. As the CTL-induced death system, the effectiveness of Fas ligand/Fas and/or Perforin/Granzyme B systems has been suggested, whereas cell death induction by CTLs was shown independently on Fas expression in the current study. Using various tetrapeptide inhibitors for caspase and its associated factor, we additionally demonstrated that inhibitors for caspase 3 (Ac-DEVD-CHO) and caspase 8/granzyme B (Ac-IETD-CHO) suppressed CTL-induced cell death, but an inhibitor for Fas-activated serine proteinase, which acts for the caspase 3 activator, did not, suggesting that CTL-induced cell death was initiated by the Perforin/Granzyme B system, rather than the Fas ligand/Fas system. On the basis of our current results, we report here that the Perforin/Granzyme B system acts dominantly for the cell death induction of HCC lines.

Many protein-free media have been developed, because protein-free media are usually more economical than serum-free or serum-containing media and facilitate the purification of bioactive materials. We evaluated various iron salts and chelating agents replacing transferrin to develop a protein-free medium for a human-human hybridoma, HB4C5, and found out that ferric citrate was favorable for the production and the productivity of monoclonal antibodies.