Sachiko Ogasawara

Aldehyde Dehydrogenase 1 Identifies Cells with Cancer Stem Cell-Like Properties in a Human Renal Cell Carcinoma Cell Line

Cancer stem cells (CSC) or cancer stem cell-like cells (CSC-LCs) have been identified in many malignant tumors. CSCs are proposed to be related with drug resistance, tumor recurrence, and metastasis and are considered as a new target for cancer treatment; however, there are only a few reports on CSCs or CSC-LCs in renal cell carcinoma (RCC). Different approaches have been reported for CSC identification, but there are no universal markers for CSC. We used two different approaches, the traditional side population (SP) approach, and the enzymatic (aldehyde dehydrogenase 1 (ALDH1)) approach to identify CSC-LC population in two RCC cell lines, ACHN and KRC/Y. We found that ACHN and KRC/Y contain 1.4% and 1.7% SP cells, respectively. ACHN SP cells showed a higher sphere forming ability, drug resistance, and a slightly higher tumorigenic ability in NOD/SCID mice than Non-SP (NSP) cells, suggesting that cells with CSC-LC properties are included in ACHN SP cells. KRC/Y SP and NSP cells showed no difference in such properties. ALDH1 activity analysis revealed that ACHN SP cells expressed a higher level of activity than NSP cells (SP vs. NSP: 32.7% vs 14.6%). Analysis of ALDH1-positive ACHN cells revealed that they have a higher sphere forming ability, self-renewal ability, tumorigenicity and express higher mRNA levels of CSC-LC property-related genes (e.g., ABC transporter genes, self-replication genes, anti-apoptosis genes, and so forth) than ALDH1-negative cells. Drug treatment or exposure to hypoxic condition induced a 2- to 3-fold increase in number of ALDH1-positive cells. In conclusion, the results suggest that the ALDH1-positive cell population rather than SP cells show CSC-LC properties in a RCC cell line, ACHN.

Overexpression of the myc target gene Mina53 in advanced renal cell carcinoma

The myc target gene Mina53 was reported to be overexpressed in esophageal cancer with a poor prognosis. The purpose of the present study was to examined Mina53 expression and its relationship to clinicopathological parameters in human renal cell carcinoma (RCC). Mina53 and Ki‐67 expression was examined on immunohistochemistry for 64 surgically resected RCC and non‐cancerous tissue. In addition, the relationship between Mina53 expression and clinicopathological prognostic factors of RCC such as age, stage, microvenous invasion (MVI), histological subtype, Ki‐67 labeling index (LI), and prognosis, was examined. Mina53 was expressed in the nuclei of tumor cells and tubular nuclei of normal renal tissue. The expression level of Mina53 was significantly higher in patients with poor prognostic factors (stage IV, MVI‐positive, and sarcomatoid RCC, and high Ki‐67 LI). The prognosis of high Mina53‐expressing tumors was significantly poorer than that of non‐Mina53‐high tumors (P < 0.0001). In conclusion, Mina53 is overexpressed in RCC tissue from patients with poor prognostic factors, suggesting that Mina53 overexpression is one of the factors for poor prognosis in RCC.

Growth inhibitory effects of pegylated IFN α‐2b on human liver cancer cells in vitro and in vivo

Abstract: Purpose: We investigated the effects of pegylated IFN‐α2b (PEG‐IFN‐α2b) on the growth of human liver cancer cells.

Expression of Fas and anti-Fas-mediated apoptosis in human hepatocellular carcinoma cell lines

BACKGROUND/AIMS Fas transduces apoptotic signals upon cross-linking with the Fas ligand, which is experimentally replaced by anti-Fas antibodies. Because little is known about Fas expression and function in hepatocellular carcinoma, these issues are addressed in the current article. METHODS We examined Fas expressions at protein and mRNA levels, and susceptibility to anti-Fas-mediated apoptosis, on six hepatocellular carcinoma cell lines. RESULTS Two cell lines constitutively expressed high levels of Fas both on their cell surface and in their cytoplasm, whereas the other four cell lines expressed Fas mainly in their cytoplasm. Fas mRNA of normal size was detected in all cell lines in reverse transcriptase-polymerase chain reaction analyses. Although a Fas mRNA variant, suggesting a soluble Fas molecule, was detected in the two cell lines expressing high levels of Fas, its amount was very small compared to that of normal-sized Fas transcript. Anti-Fas dose-dependently induced apoptosis exclusively in the two cell lines which constitutively express high levels of cell surface Fas. However, after preincubation with interferon-gamma, one cell line with low surface Fas expression became anti-Fas sensitive equivalent to the two cell lines expressing surface Fas at high levels. Studies of two clonally related cell lines showed that dedifferentiated clones had lower Fas expression and resistance to anti-Fas, suggesting deterioration of Fas system after clonal cell dedifferentiation. CONCLUSIONS These findings suggest sensitivity to anti-Fas is virtually relevant to cell surface Fas, but not to cytoplasmic Fas expression. However, its expression level does not correlate to sensitivity to anti-Fas.

Prostatic acid phosphatase as a target molecule in specific immunotherapy for patients with nonprostate adenocarcinoma.

Prostatic acid phosphatase (PAP) is one of the prostate-related antigens that are applicable to specific immunotherapy for patients with prostate cancer. In this study, we determined whether or not PAP could be a target molecule in specific immunotherapy for patients with nonprostate cancer. A variety of adenocarcinoma cell lines were examined for their PAP expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blot analysis, respectively. Considerable percentages of colon, gastric, and breast cancer cell lines were found to be positive for PAP at both the mRNA and the protein levels. The PAP expression in cancer tissues was also confirmed by immunohistochemical staining. In addition, we examined whether cancer-reactive cytotoxic T lymphocytes (CTLs) could be induced from peripheral blood mononuclear cells (PBMCs) of human leukocyte antigen (HLA) A24+ nonprostate cancer patients by in vitro stimulation with a PAP peptide. As a result, tumor-specific CTLs could be induced from the PBMCs of HLA-A24+ colon and gastric cancer patients. Their cytotoxicity against HLA-A24+ cancer cells was dependent on PAP peptide-specific and CD8+ T cells. These findings indicate that PAP could be a target molecule in specific immunotherapy for patients with nonprostate adenocarcinomas including colon and gastric cancers.

Hepatitis C virus RNA present in saliva but absent in breast‐milk of the hepatitis C carrier mother

In order to examine whether saliva and breast‐milk are mediators of the vertical transmission of hepatitis C virus (HCV) from an HCV carrier mother, serum, saliva, and breast‐milk samples from 11 HCV carrier mothers were collected at the time of delivery, and at approximately 1‐ to 3‐month intervals for as long as 30 months postpartum. Serum was also sampled from their children. All samples were analysed for the presence of HCV RNA, using the nested polymerase chain reaction method. No HCV RNA was detected in any breast‐milk samples. In saliva, HCV RNA was detected in four of the 11 mothers (36%). These four mothers also had liver function abnormalities. Hepatitis C virus RNA was not detected in any serum samples of the children, and all children had normal liver function. The children were monitored for periods from 2 to 44 months. During this period, there was no evidence of virus transmission. Breast‐milk is not likely to be a source of mother‐to‐child transmission of HCV. Maternal saliva may harbour HCV, but it may not result in infant infection.

Expression of angiogenic factors, basic fibroblast growth factor and vascular endothelial growth factor, in human biliary tract carcinoma cell lines

In order to clarify angiogenic mechanism in biliary tract carcinoma, expressions and functions of basic fibroblast growth factor (bFGF) and its receptors (FGFR-1-4), and vascular endothelial growth factor (VEGF) and its receptors were investigated by using human biliary tract carcinoma cell lines (KMC-1, KMC-2, KMBC and KMG-C). Expression of bFGF was confirmed in KMC-1 and KMC-2, and that of FGFR-1-4 in all the cell lines except no FGFR-2 in KMC-2. Expression of VEGF was detected in all the cell lines, whereas the cell lines did not express VEGF receptors. Addition of anti-bFGF neutralizing antibody to the medium did not suppress cell proliferation, whereas exogenous bFGF with or without heparin accelerated cell proliferation in all cell lines. Addition of anti-bFGF neutralizing antibody or anti-VEGF neutralizing antibody to the co-culture of human umbilical vascular endothelial cells (HUVEC) and KMC-2 suppressed the proliferation of HUVEC. Surgically obtained cholangiocarcinoma tissues (n=7) were immunohistochemically negative to bFGF, while six of the seven were positive to VEGF. These findings suggested that human biliary tract carcinoma cells express both bFGF and VEGF not as autocrine growth factors but as angiogenic factors. On the other hand, expression of VEGF was found at a higher frequency than bFGF both in the cell lines and tissues.

Accelerated expression of a Myc target gene Mina53 in aggressive hepatocellular carcinoma

Aim:  Expressions of the myc target genes Mina53 and mimitin are high in esophageal squamous cell carcinoma and colon cancer, and their relationship to cell proliferation and patient prognosis has been reported. Because c‐myc gene expression is closely related to hepatocellular carcinoma (HCC) growth or formation and/or maintenance, we examined the Mina53 and mimitin expressions in HCC.

Growth inhibitory effects of interferon-α subtypes vary according to human liver cancer cell lines

Background:  Interferon (IFN)‐α preparations used in the treatment of viral and neoplastic disease consist of single or multiple IFN‐α subtypes that may possess different biological activity, but there are no data on liver cancer cells.

N-myc downstream regulated gene1/Cap43 overexpression suppresses tumor growth by hepatic cancer cells through cell cycle arrest at the G0/G1 phase

N-myc downstream regulated gene-1 (NDRG1)/Cap43 regulates tumor growth and metastasis in various carcinomas. In this study we examined whether and how NDRG1/Cap43 modulates tumor growth by human hepatocellular carcinoma (HCC) cells. NDRG1/Cap43 cDNA was used to transfect HCC cell lines (KIM-1), and stable transfectants overexpressing NDRG1/Cap43 (KIM-1/Cap43) were obtained. Cell cycle analysis showed that KIM-1/Cap43 cells were arrested in the G0/G1 phase. Western blot analysis demonstrated an increase in p21 in KIM-1/Cap43 cells in culture under full confluency as compared with KIM-1/Mock. When KIM-1 cells, which are very low in NDRG1/Cap43 expression, were treated with mimosine, a G0/G1 cell cycle blocker, expression of NDRG1/Cap43 was induced in a dose dependent manner, together with p21 induction and CDK4 reduction. In vivo, KIM-1/Cap43 cells showed markedly decreased tumor growth rates compared with those of KIM-1/Mock. Immunohistochemical staining demonstrated markedly higher p21 labeling index in the KIM-1/Cap43 tumor than KIM-1/Mock tumor, and lower CDK4 and Ki-67 labeling index in the KIM-1/Cap43 than KIM-1/Mock. In order to confirm suppressive effects of NDRG1/Cap43, we further established a stable transfectant expressing NDRG1/Cap43 (HAK-1B/Cap43) using another HCC cell line, HAK-1B. Western blot analysis demonstrated an increase in p21 and a decrease in CDK4 in HAK-1B/Cap43 cells in culture under full confluency as compared with HAK-1B/Mock. HAK-1B/Cap43 also showed decreased tumor growth rates as compared with its control counterpart in vivo. NDRG1/Cap43 overexpression thus induced cell cycle arrest at the G0/G1 phase accompanied by increased p21 and decreased CDK4 expression in HCC cells. NDRG1/Cap43 might play a key role in the cell cycle control of G0/G1 in HCC cells.