Koichi Asanuma

Survivin as a Radioresistance Factor in Pancreatic Cancer

We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription‐polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC‐1 cells, which had the highest survivin mRNA levels, were most resistant to X‐irradiation; MIAPaCa‐2 cells, which showed the least survivin mRNA expression, were the most sensitive to X‐irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC‐1 and MIAPaCa‐2 cells were subjected to sublethal doses of X‐irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC‐1 and MIAPaCa‐2 cell lines by pretreatment with a sublethal dose of X‐irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase‐3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy.

Detection of Anti-Livin Antibody in Gastrointestinal Cancer Patients

Livin, a recently described member of the inhibitor of apoptosis protein (IAP) family, contains a single baculovirus IAP repeat and a carboxyl-terminal RING finger (1)(2)(3). Like other proteins in the IAP family, livin binds specifically to a terminal effector cell-death protease, in this instance, caspase-9 (1)(2)(3). The consequences are substantially reduced caspase activity and reduced cell death in response to diverse apoptotic stimuli (1)(2)(3). Semiquantitative reverse transcription-PCR methods have detected human livin mRNA in fetal kidney, heart, and spleen and in adult tissues such as heart, lung, spleen, ovary, and placenta (4). In addition, livin mRNA is overexpressed by some cancer cells, including melanoma, breast cancer, cervical cancer, colon cancer, prostate cancer, leukemia, and lymphoma cells (4). As with survivin overexpression (5)(6)(7), livin overexpression by cancer cells may lead to anti-livin antibody responses and cytotoxic T-lymphocyte responses against the cancer. In the present study, we examined livin mRNA expression in gastrointestinal cancer cell lines and the prevalence of antibody responses against livin in patients with various gastrointestinal cancers. We cultured human pancreatic cancer cell lines (PANC-1, Capan-1, AsPC-1, MIAPaCa-2, and BxPC-3), gastric cancer cell lines (MKN-1, MKN-45, and TMK-1), colon cancer cell lines (HT-29, SW480, SW620, and LSI180), and a hepatoma cell line (HepG2) in RPMI-1640 with 100 mL/L calf serum at 37 °C in 5% CO2. Livin mRNA expression was quantified by a previously reported method (8). The sequence of the forward primer was 5′-TCAGTTCCTGCTCCGGTCA-3′, that the reverse primer was 5′-CGTCTTCCGGTTCTTCCCA-3′, and the sequence of the TaqMan probe was 5′-CCACAGTGTGCAGGAGACTCACTCCC-3′. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was used and amplified with TaqMan control reagents (Perkin-Elmer Applied Biosystems). The conditions of the one-step reverse transcription-PCR have been described previously …

A Role for Survivin in Radioresistance of Pancreatic Cancer Cells

Using gene‐transduced pancreatic cancer cells, we examined whether survivin expression is directly involved in regulation of radiosensitivity. Ordinarily radiosensitive MIAPaCa‐2 cells transduced with wild‐type survivin gene (MS cells) proliferated more rapidly than cells transduced with control vector. MS cells were significantly less radiosensitive than control vector‐transduced cells. Radiation‐induced activity of caspase‐3, but not caspase‐7, was significantly inhibited in MS cells. On the other hand, transduction of a dominant‐negative mutant survivin gene into radioresistant PANC‐1 cells augmented radiosensitivity. Further, the radiation‐induced increase in caspase‐3 activity was enhanced, indicating that survivin function was truly inhibited. These results indicate that survivin expression directly down‐regulates radiosensitivity.

Survivin Enhances Fas Ligand Expression via Up-Regulation of Specificity Protein 1-Mediated Gene Transcription in Colon Cancer Cells

Cancer cells are thought to possess mechanisms for evading the host’s immune surveillance system. Survivin, a member of the inhibitor-of-apoptosis family overexpressed by cancer cells, inhibits Fas-mediated apoptosis induced by immune cells. In addition, cancer cells express Fas ligand (FasL) on their surfaces as a counterattack against immune cells. Mechanisms by which cancer cells express FasL, including involvement of survivin, are unclear. In the present study, we demonstrated that survivin up-regulated FasL expression and investigated how this might occur. Quantitative immunostaining showed correlation between survivin and FasL protein expression in colon cancer tissues (r = 0.79). FasL expression was up-regulated in LS180 colon cancer cells transfected with the survivin gene. Transfectants showed increased cytotoxicity against a Fas-sensitive human T leukemia cell line, Jurkat. In contrast, FasL expression was down-regulated in SW480 cells transfected with a small inhibitory RNA to prevent survivin expression. Survivin gene transfectants showed increased DNA binding of transcription factor specificity protein 1 (Sp1) to the FasL promoter, and up-regulation of Sp1 phosphorylation at serine and threonine residues; the total amount of Sp1 was unchanged. Thus, survivin enables cancer cells not only to suppress immune cell attack by inhibiting Fas-mediated apoptotic signaling, but to attack immune cells by induction of FasL.

Survivin as a predictor of cis-diamminedichloroplatinum sensitivity in gastric cancer patients.

Survivin, a member of the inhibitor‐of‐apoptosis family, inhibits apoptosis by blocking caspase‐3 and ‐7 activation. Gastric cancer, which is among the most intractable of malignant tumors, is known for resistance to various drugs, including cis‐diamminedichloroplatinum (CDDP). Since this agent induces apoptosis via caspase‐3 activation, survivin may mediate the drug resistance. We investigated survivin messenger RNA (mRNA) expression in gastric cancers and the relationship between expression and sensitivity to CDDP. Expression of the survivin gene was significantly up‐regulated in gastric cancers compared to the tissues of normal mucosa, atrophic gastritis, and intestinal metaplasia (P<0.0001) as assessed by a quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), and was negatively associated with overall survival of patients who received CDDP‐based chemotherapy. To investigate whether survivin is a resistance factor against CDDP‐induced apoptosis, we transfected wild‐type and dominant‐negative mutants of the survivin gene into gastric cancer cells using a lipofection method. Overexpression of survivin protected MKN45 cells from CDDP‐induced apoptosis. Expression of the dominant‐negative mutant of the survivin gene sensitized NUGC‐3 cells to drug‐induced apoptosis. These results indicate that survivin may be pivotal in the development of gastric cancer and resistance to CDDP, and therefore controlling expression of the survivin gene may be therapeutically useful. (Cancer Sci 2004; 95: 44–51)

Survivin Down-regulation Plays a Crucial Role in 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitor-induced Apoptosis in Cancer

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-XL, revealed that only survivin expression was decreased by lovastatin. Survivin down-regulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin down-regulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.

Mutations of the p53 Gene and Loss of Heterozygosity at Chromosome 17p13.1 are Associated with Increased Survivin Expression in Breast Cancer

Expression of survivin, a member of the inhibitor-of-apoptosis (IAP) family, is elevated in fetal tissues and in various human cancers originating in the breast, lung, prostate, colon, pancreas, and stomach. Since overexpression of the survivin gene has been linked to poor patient survival in several cancers, survivin may be an important prognostic marker. Mechanisms up-regulating survivin gene expression in cancer are poorly understood. Recently, wild-type p53 was found to repress expression of the survivin gene by binding to the survivin promoter, thereby inhibiting promoter activity. Further, loss of heterozygosity (LOH) at 17p13 distal to the p53 gene is associated with more aggressive behavior of breast cancers. We therefore tested the hypothesis that not only p53 gene mutation but also LOH at 17p13 can up-regulate survivin expression in breast cancer. Survivin mRNA expression was greater in cancers than in uninvolved tissues (p < 0.0001). Mutations of the p53 gene were detected in 5 of 25 tumors; higher survivin gene expression was evident in these. LOH at the D17S938 locus (17p13.1) was found in 10 of 25 tumors, and most of these also showed increased survivin gene expression. Thus expression of survivin may be regulated not only by p53 but additionally by a putative tumor suppressor gene located at 17p13 distal to the p53 gene.

Downregulation of cytokine-induced neutrophil chemoattractant and prolongation of rat liver allograft survival by interleukin-10.

We investigated whether the administration of recombinant human interleukin-10 (rhIL-10) regulates the production of cytokine-induced neutrophil chemoattractant (CINC) and improves graft survival in rat orthotopic liver transplantation (OLTx). Allograft recipients received injections of rhIL-10 at doses of 2, 10, 20, or 50 micrograms/kg/day. The allograft recipients that received rhIL-10 at 10 or 20 micrograms/kg/day showed a slight but significant prolongation of graft survival to 13.0 +/- 0.4 and 13.8 +/- 0.3 days, respectively, compared with 9.6 +/- 0.2 days in untreated allografts. Conversely, the administration of high-dose rhIL-10 shortened the allograft survival. In the rhIL-10 treatment groups, the mean serum and tissue levels of CINC at every time point after OLTx were reduced significantly compared with those in the no-treatment group. The mean peak neutrophil counts in the peripheral circulation (PC) of the groups given rhIL-10 at 10, 20, or 50 micrograms/kg/day from the samples obtained 12h after reperfusion were decreased significantly compared with the no-treatment group. Furthermore, the mean peak neutrophil counts in the PC of the groups given rhIL-10 at 10 or 20 micrograms/kg/day from the samples obtained between postoperative days (PODs) 7 and 10 were decreased significantly compared with the no-treatment group. The magnitude of liver damage and leukocyte infiltration in the rhIL-10-treated allografts on PODs 1 and 7 was reduced compared with that of untreated allografts. Our data indicate that the administration of rhIL-10 downregulates CINC production during the period of reperfusion injury and acute cellular rejection after OLTx, and prolongs liver allograft survival, suggesting that IL-10 therapy is potentially beneficial in OLTx.

Prevalence of human anti-mouse antibodies (HAMAs) in routine examinations.

BACKGROUND Circulating heterophilic antibodies interfere with immunological assays in laboratory examinations; however, their rate of incidence is currently questionable. We developed an enzyme-linked immunosorbent assay (ELISA) to detect human anti-mouse antibodies (HAMAs) in routine examinations. METHODS The study samples were comprised of serum samples obtained from 290 inpatients and outpatients at our hospital. Mouse immunoglobulin G1 (mIgG1), mIgG2a, and mIgG2b were used as the antigens and horseradish peroxidase (HRP)-conjugated anti-human IgG and IgM were used to identify the HAMA isotype. RESULTS HAMAs were detected in 11.7% (34/290) of the samples. We observed 18 and 20 samples positive for IgG- and IgM-type HAMAs, respectively. Four samples contained both IgG- and IgM-type HAMAs. HAMAs against mIgG1, mIgG2a, and mIgG2b were found in 21, 14, and 13 samples, respectively. Existence of HAMAs was confirmed by western blotting using mIgGs as the antigens and HAMAs as the primary antibodies. Heterophilic blocking reagent (HBR) was also used to block the heterophilic interactions. Unexpectedly, a low HBR concentration rather enhanced the interactions instead of blocking them. CONCLUSIONS A considerable number of HAMA-positive samples, reacting with the heavy chain of mIg, were found in routine examinations. A sufficient amount of HBR should be used for blocking the heterophilic interactions.

Role of Human Telomerase Reverse Transcriptase and Telomeric‐repeat Binding Factor Proteins 1 and 2 in Human Hematopoietic Cells

Telomerase, an enzyme that adds hexameric repeats of 5′‐TTAGGG‐3′, termed telomeres, to the ends of chromosomal DNA, has been implicated in cellular immortalization and cellular senescence. Recently several relevant genes have been cloned, including those encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase‐associated protein‐1 (TEP1). Also important are genes encoding human telomeric‐repeat binding factor proteins (TRF) 1 and 2. We compared 10 human malignant hematopoietic cell lines, 19 samples from patients with acute leukemia and normal granulocytes and monocytes to study telomerase activity and expression of these various genes using a reverse transcription‐polymerase chain reaction (RT‐PCR). In all 10 malignant cell lines with telomerase activity, hTR, hTERT mRNA, and TEP1 mRNA were expressed, while in normal monocytes and granulocytes without telomerase activity, expression of hTR, but not hTERT mRNA was detected. TEP1 mRNA was expressed in normal monocytes, but not granulocytes. Expression of TRF1 and TRF2 mRNAs was greater in the normal cells than in human malignant hematopoietic cell lines and in 16 samples of patients with acute leukemia. When differentiation of the malignant hematopoietic cell line HL‐60 was induced using tumor‐necrosis‐factor 471 and all‐trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation. Of the three telomerase components, only hTERT mRNA expression showed changes paralleling telomerase activity, becoming undetectable with differentiation. In contrast, initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. Not only hTERT, but also TRF1 and TRF2 are important regulators of telomerase activity that represent potential targets for gene therapy against cancer.