GLI1 interferes with the DNA mismatch repair system in pancreatic cancer through BHLHE41-mediated suppression of MLH1.

The mismatch repair (MMR) system is indispensable for the fidelity of DNA replication, the impairment of which predisposes to the development and progression of many types of cancers. To date, GLI1 transcription factor, a key molecule of the Hedgehog signaling pathway, has been shown to regulate the expression of several genes crucial for a variety of cancer cell properties in many types of cancers, including pancreatic ductal adenocarcinoma (PDAC), but whether GLI1 could control the MMR system was not known. Here, we showed that GLI1 and GLI2 indirectly suppressed the expression of MLH1 in PDAC cells. Through GLI1 target gene screening, we found that GLI1 and GLI2 activated the expression of a basic helix-loop-helix type suppressor BHLHE41/DEC2/SHARP1 through a GLI-binding site in the promoter. Consistent with a previous report that BHLHE41 suppresses the MLH1 promoter activity, we found that the activation of GLI1 led to the BHLHE41-dependent suppression of MLH1, and a double knockdown of GLI1 and GLI2 conversely increased the MLH1 protein in PDAC cells. Using TALEN-based modification of the MLH1 gene, we further showed that GLI1 expression was indeed associated with an increased tolerance to a methylating agent, methylnitrosourea cooperatively with a lower copy number status of MLH1. Finally, GLI1 expression was immunohistochemically related positively with BHLHE41 and inversely with MLH1 in PDAC cells and precancerous lesions of the pancreas. On the basis of these results, we propose that GLI1 depresses the MMR activity and might contribute to the development and progression of PDAC.

A new molecular targeted therapeutic approach for renal cell carcinoma with a p16 functional peptide using a novel transporter system.

Molecular targeting agents have become formidable anticancer weapons showing much promise against refractory tumors and functional peptides and are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms the basis for a potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. We examine the growth suppression efficiency of human renal cell carcinoma (RCC) by single-peptide targeting. We simultaneously introduced p16INK4a tumor suppressor peptides by Wr-T-mediated peptide delivery. Wr-T-mediated transport of p16INK4a functional peptide into 10 RCC lines, lacking expression of the p16INK4a molecule, reversed the specific loss of p16 function, thereby drastically inhibiting tumor growth in all but 3 lines by >95% within the first 96 h. In vivo analysis using SK-RC-7 RCC xenografts in nude mice demonstrated tumor growth inhibition by the p16INK4a peptide alone, however, inoculation of Wr-T and the p16INK4a functional peptide mixture, via the heart resulted in complete tumor regression. Thus, restoration of tumor suppressor function with Wr-T peptide delivery represents a powerful approach, with mechanistic implications for the development of efficacious molecular targeting therapeutics against intractable RCC.

Detection of elevated proteins in peritoneal dissemination of gastric cancer by analyzing mass spectra data of serum proteins.

BACKGROUND: Recently, several authors reported on the Protein Chip approach to analyze serum. They used SELDI-TOF-MS (surface enhanced laser desorption/ionization-time of flight-mass spectrometry) to identify patients with cancers of various origins in a highly sensitive and specific manner. In the current study, a similar approach was employed to analyze the serum of patients with various stages of gastric cancer. METHODS: Control serum specimens from patients with gastritis (n = 19) and those with gastric cancer (Stage I: n = 6, Stage II or III: n = 6, Stage IV: n = 6, total: n = 18) were collected and analyzed by the Protein Chip biomarker system (Bio-Rad, Japan), a platform for SELDI-TOF-MS, and protein profiles were obtained and compared. The cation exchange chip (CM10) and the anion exchange chip (Q10) were used for processing before TOF-MS. RESULTS: nine proteins were significantly over-expressed (P < 0.05, Student t-test) in patients with gastric cancer compared to patients with gastritis. Among them, four protein masses with 2929 m/z, 3293 m/z, 3371 m/z, and 4213 m/z were found to be differentially expressed solely in patients suffering from peritoneal dissemination. All peaks were processed on CM10 chips. Employing one data mining method, CART (classification and regression trees), gastric cancer patients with peritoneal dissemination were successfully separated from those who had no peritoneal seeding. CONCLUSION: A validation study with a larger number of samples is mandatory; however, the detected peaks here might be candidates for biomarkers of peritoneal dissemination and/or gastric cancer. Moreover, further analysis of these four proteins might be helpful in revealing the mechanism of peritoneal dissemination, which at present has no cure.

Pleomorphic carcinoma with osteoclastic giant cells of the breast: immunohistochemical differentiation between coexisting neoplastic and reactive giant cells.

Herein is described a unique case of breast carcinoma with two different types of giant cells noted in both cytological and histological specimens. A 51-year-old Japanese woman noticed a hard mass in the upper outer quadrant of her left breast. Aspiration cytology exhibited numerous anaplastic giant cells; the cytological diagnosis was high-grade ductal carcinoma, although a few osteoclastic giant cells were also observed. A left simple mastectomy and sentinel lymph node biopsy were performed. Histologically, approximately 90% of the tumor was composed of giant cells; conventional invasive ductal carcinoma and ductal carcinoma in situ were found focally at the periphery of the tumor. The main part of the tumor contained both anaplastic, neoplastic giant cells and non-neoplastic, osteoclastic giant cells that were distinguishable from nuclear atypism. The presence of the two types of giant cells was also confirmed on immunohistochemistry using a histiocytic marker (CD68) and two epithelial markers (AE1/AE3 and CAM5.2). Based on the latest World Health Organization classification, the diagnosis was pleomorphic carcinoma with osteoclastic giant cells. To the authors' knowledge there has been no previous report on this subject except for a single case mentioned in Rosen's Breast Pathology.

Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells.

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0-3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.

Combinations of tumor-specific CD8+ CTLs and anti-CD25 mAb provide improved immunotherapy.

One new approach to cancer therapy is based on the adoptive transfer of tumor-specific cytotoxic T cells and anti-CD25 antibodies. In the present study, CD8+ and IFN-gamma secreting T lymphocytes (CTLs) were enriched as tumor-specific cytotoxic T cells from spleen lymphocytes of mice bearing the Renca tumor (a murine renal carcinoma line originating from a BALB/c mouse) after stimulation with tumor cells. An anti-CD25 IL-2Ralpha(anti-CD25) mAb from hybridoma PC61 was used for depletion for CD4(+)CD25(+) regulatory T (Treg) cells. Treatment-efficacy for tumor-bearing mice was compared using 4 systems: 1, whole spleen lymphocytes stimulated with tumor cells in vitro from tumor-bearing mice; 2, CTLs; 3, anti-CD25 mAbs; 4, CTLs and anti-CD25 mAbs. At the 50th day after tumor inoculation, in the group which received anti-CD25 mAb for depletion of T cells and inoculation of CTLs, tumors had disappeared and no re-growth was observed. In contrast, all mice of the non-treated and other three groups, treated with whole spleen cells alone, CTLs alone and anti-CD25 mAb alone, had died. These results showed that a combination of Treg cell-depletion using anti-CD25 mAbs and CTL administration is a feasible approach for treatment of cancers which warrants further exploration in the clinical setting.

Prognostic significance of immunohistochemical expression of the HER-2/neu oncoprotein in bone metastatic prostate cancer.

OBJECTIVES: To investigate the usefulness of the overexpression of the human epidermal growth factor receptor (HER-2) oncoprotein in patients with bone metastatic prostate cancer as a marker for the time to recurrence and outcome after endocrine therapy. METHODS: We studied 50 patients who had been diagnosed with bone metastatic prostate cancer. HER-2 overexpression in the prostatic tissue by biopsy was evaluated by immunohistochemistry using the Hercep test. The results were scored into four levels by two pathologists; scores greater than 1+ were considered positive. RESULTS: The HER-2 staining score was 0, 1+, 2+, 3+, and indeterminate in 28, 4, 11, 6, and 1 case, respectively. HER-2 was overexpressed (greater than 1+) in 21 patients (42%). The cause-specific survival and nonrecurrence rates were significantly lower in the HER-2-positive group than in the negative group (P = 0.0084 and P = 0.0485, respectively). Furthermore, the cause-specific survival rate after recurrence was significantly greater in the HER-2-negative group than in the positive group (P = 0.0247). CONCLUSIONS: We consider that HER-2 overexpression, as measured by immunohistochemistry, may be useful as a marker of an unfavorable prognosis by predicting the interval until relapse and outcome after endocrine therapy in patients with bone metastatic prostate cancer.

Differential profiling analysis of proteins involved in anti-proliferative effect of interferon-alpha on renal cell carcinoma cell lines by protein biochip technology.

Interferon-alpha (IFN) is widely used for the treatment of progressive renal cell carcinoma (RCC), but its effective response rate is only about 15%. New biomarkers of RCC contributing to the effective IFN treatment are needed to establish a sensitivity test for evaluating if the IFN is effective or not against RCC. All the proteins expressed in the IFN-susceptible and -resistant RCC cell lysates were analyzed by surface enhanced laser desorption ionization (SELDI) mass spectrometry using ProteinChip technology and their different protein expression were detected by the comparison of the profiles between them. We detected the following candidate markers that exhibited peak shifts: a) in the IFN-susceptible cell lines, a candidate marker with a molecular weight of 5,688 Da was detected on a hydrophobic (H4) chip: b) in the IFN-resistant cell lines, candidate markers each with a molecular weight of 8,049, 3,157, 3,993, and 8,959 Da were detected on strong anion exchange (SAX2, pH 9.0, two types) chips, H4 chip, and weak cation exchange (WCX, pH 9.0) chips, respectively. IFN treatment produced no weight increase in these four proteins, and c) candidate marker with a molecular weight of 1,623 Da that was expressed in both cell lines after the IFN treatment was detected on the H4 chip. These data suggest that the ProteinChip system is very useful in identifying proteins showing unique peaks in the RCC cell lines with different IFN susceptibility, and the comparison of these proteins measured in RCC cell lysates may help to identify the IFN sensitivity. Furthermore, the discovery of a susceptibility and or a inhibitory factor may eventually lead to the development of a novel drug targeting the respective factor for the improvement of anticancer chemotherapy.

Functional characterization of 3 thioredoxin homology domains of ERp72.

Folding of secretory proteins is associated with the formation and isomerization of disulfide bonds. ERp72, a protein disulfide isomerase (PDI) family member, possesses 3 thioredoxin homology domains, but the participation of each domain in disulfide-bond formation and isomerization remains to be determined. We analyzed the function of individual domains in the insulin reduction assay system by site-directed mutagenesis with cysteine-to-serine replacement. All domains contributed to apparent steady-state binding (Km) and catalysis at saturating substrate concentrations (kcat) but in different manners. A mutant ERp72 with mutations in domains 1 and 2 (ERp72-mut-1+2) exhibited reductions in kcat of 73.9% when compared with wild type, whereas ERp72-mut-1+3 (mutations in domains 1 and 3) and ERp72-mut-2+3 (mutations in domains 2 and 3) exhibited less substantial reductions in kcat. ERp72-mut-1+3 and ERp72-mut-2+3 showed elevations in Km of 89.9% and 96.2%, respectively, when compared with wild type, whereas ERp72-mut-1+2 exhibited smaller elevations in Km. These results suggest that domains 1 and 2 make greater contributions to catalyzing efficacy and domain 3 to binding affinity. Domain 2 is involved in binding affinity, in combination with domain 3, in addition to its own contribution to catalyzing efficacy. This assignment of functions to individual domains is similar to that observed in other PDI domains, which is consistent with the high sequence homology between ERp and PDI domains.

Differential cooperative enzymatic activities of protein disulfide isomerase family in protein folding.

Endoplasmic reticulum (ER)p61, ERp72, and protein disulfide isomerase (PDI), which are members of the PDI family protein, are ubiquitously present in mammalian cells and are thought to participate in disulfide bond formation and isomerization. However, why the 3 different members need to be colocalized in the ER remains an enigma. We hypothesized that each PDI family protein might have different modes of enzymatic activity in disulfide bond formation and isomerization. We purified PDI, ERp61, and ERp72 proteins from rat liver microsomes and compared the effects of each protein on the folding of bovine pancreatic trypsin inhibitor (BPTI). ERp61 and ERp72 accelerated the initial steps more efficiently than did PDI. ERp61 and ERp72, however, accelerated the rate-limiting step less efficiently than did PDI. PDI or ERp72 did not impede the folding of BPTI by each other but rather catalyzed the folding reaction cooperatively with each other. These data suggest that differential enzymatic activities of ERp proteins and PDI represent a complementary contribution of these enzymes to protein folding in the ER.

Identification of specific autoantigens in Sjögren's syndrome by SEREX.

We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjögren's syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in the sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch-like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A markedly high frequency of anti-IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS-B/La. The presence of autoantibodies against KLHL12 and KLHL7 in the sera was significantly specific to SjS (23% and 17%, respectively), as they were not detected in RA, SLE or HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in the salivary glands and immuno-privileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ-specific autoantigens in the aetiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with anti-SS-B/La autoantibodies.

Immunohistochemical study of chromogranin A in Stage D2 prostate cancer.

OBJECTIVES: To study the clinical and pathologic factors that affect the time to recurrence after hormonal therapy in patients with Stage D2 prostate cancer and to determine whether cells positive for chromogranin A (CgA) in tissue biopsied at diagnosis is a useful marker for predicting the duration to recurrence after hormonal therapy. METHODS: A total of 50 patients diagnosed with Stage D2 prostate cancer at our institution from January 1998 to December 2001 were studied. The needle prostate biopsy specimens obtained at diagnosis were stained by immunohistochemistry using the labeled streptavidin biotin method, and the results were considered positive if CgA-positive cells constituted 10% or more of the tumor area. RESULTS: No significant differences were found between patients on the basis of the time to recurrence of less than 2 years versus 2 years or more with respect to prostate-specific antigen level, patient age, Gleason score, or extent-of-disease grade. Of the 50 patients, 11 (22.0%) had positive cell staining for CgA. No significant differences were found between the CgA-positive group and the CgA-negative group in age, prostate-specific antigen level, Gleason score, extent-of-disease grade, T stage, or N stage. The rate of freedom from recurrence after 2 years as analyzed by the Kaplan-Meier method was 18.2% for the CgA-positive group and 47.4% for the CgA-negative group, and the CgA-positive group had a significantly shorter time to recurrence (P < 0.0122). CONCLUSIONS: The results of our study have shown that patients with overexpression of CgA-positive cells in the prostate have a significantly shorter time to recurrence after hormonal therapy than patients with CgA-negative cells; thus, CgA expression may be a useful marker predictive of the time to recurrence.

Production and characterization of an active single-chain variable fragment antibody recognizing CD25.

The alpha subunit of the interleukin-2 receptor (IL-2Ralpha, CD25) is a potential target in therapeutic approaches for hematolopoietic malignancies expressing CD25 on their cell surface, such as adult T cell leukemia/lymphomas. Recent reports have demonstrated that depletion of CD4+CD25+ regulatory T cells with anti-CD25 antibodies may enhance host tumor immunity. We previously raised a mouse monoclonal antibody (mAb), Ta60b mAb (IgG1kappa), specifically recognizing CD25, and an attempt was made here to produce a single chain Fv fragment (scFv) from this mAb as an initial step to development of scFv-based therapeutics. cDNA fragments encoding for the variable regions of the light and heavy chains of the Ta60b mAb were thus isolated by polymerase chain reaction-mediated cloning, and, an expression vector constructed to express Ta60b scFv fused with the maltose binding protein (MBP) in the periplasm of Escherichia coli. The soluble form of MBP-Ta60b fused scFv could be extracted and affinity-purified with an amylose/agarose column, allowing its immunoreactivity to be analyzed by enzyme-linked immunosorbent assay (ELISA), mixed hemadsorption assay, and fluorescence activated cell sorting. In addition, binding activity was studied by competitive ELISA and surface plasmon resonance. The results showed that Ta60b scFv obtained from periplasm retains good reactivity, although its KD value was 4-fold lower than that of the whole Ta60b antibody, suggesting possible clinical use for treatment of patients with CD25-expressing tumors and also for enhancing anti-tumor immunity.

The level of cadherin-6 mRNA in peripheral blood is associated with the site of metastasis and with the subsequent occurrence of metastases in renal cell carcinoma.

BACKGROUND: To evaluate the significance of the presence of circulating renal cell carcinoma (RCC) cells in the development of metastases, the authors extended a previous study to quantify cadherin-6 mRNA levels in association with the pattern of metastasis. METHODS: Cadherin-6 mRNA levels were measured in peripheral blood samples from 66 patients with RCC, including 55 patients who had newly diagnosed RCC (43 without metastases and 12 with metastases) and 11 patients who had recurrent RCC. For quantitative polymerase chain reaction analysis, a cutoff value was determined in blood samples from 25 healthy volunteers and was verified in samples from 5 healthy controls and from 10 patients who had other malignancies. The correlation between the site of metastases and the cadherin-6 mRNA level was analyzed, and a follow-up study (median, 39 months) to track subsequent metastases was performed after patients underwent nephrectomy. RESULTS: Cadherin-6 was found in 69.9% of patients with metastases and in 34.9% of patients without apparent metastases (P = 0.0099). In the group of patients with recurrent RCC, patients who had only pulmonary metastases had a significantly lower positivity rate (25.0%) compared with patients who had distant metastases (85.7%; P = 0.044). Among 43 patients with newly diagnosed RCC, 5 of 15 patients who were positive for cadherin-6 had metastases after nephrectomy, whereas only 2 of the 28 patients with negative cadherin-6 status had recurrent disease (P = 0.0398). In addition, the recurrence-free survival of patients who were positive for cadherin-6 was poorer compared with the survival of patients who were negative for cadherin-6 (P = 0.062). CONCLUSIONS: The quantification of cadherin-6 mRNA in peripheral blood may be a significant predictive marker for current and future metastases. However, subsequent metastases did not always correlate with levels of cadherin-6 mRNA. This may have been due either to the small numbers of circulating tumor cells or to the low levels cadherin-6 mRNA in circulating tumor cells.

A role of activated Sonic hedgehog signaling for the cellular proliferation of oral squamous cell carcinoma cell line.

Sonic hedgehog (Shh) is a secreted morphogen crucial for appropriate cellular proliferation during mammalian development. The activated Shh signaling is known to predispose to human tumors such as medulloblastoma and basal cell carcinoma, while a role of Shh signaling in the other common tumors is still controversial. Here we showed the overexpression of Shh in five cell lines among 14 human oral squamous cell carcinoma (OSCC) cell lines. One of the Shh-expressing OSCC cell lines HSQ-89 showed the inhibition of G1/S transition and apoptotic cell death by treatment with Cyclopamine, a steroidal alkaloid that blocks the intracellular Shh signaling. Furthermore, we found that treatment with Y-27632, a specific inhibitor of Rho-associated kinase, mimicked the effect of Cyclopamine on the cell cycle progression of HSQ-89. Our study revealed the involvement of activated Shh signaling in the cellular proliferation of OSCC cells, indicating Shh signaling might be a good therapeutic target for OSCC.

Detection of cadherin-6 mRNA by nested RT-PCR as a potential marker for circulating cancer cells in renal cell carcinoma.

To detect circulating RCC cells, we established a nested RT-PCR system for cadherin-6 mRNA, which is specifically expressed in RCC. A total of 121 samples of peripheral blood (34 healthy volunteers and 87 patients with RCC) were analyzed in this study. Total RNA of the monocyte fraction of the blood was extracted, then nested RT-PCR using specific primers was performed to detect mRNA of N-cadherin or cadherin-6. Nested RT-PCR revealed that expression of cadherin-6 mRNA was not present in the blood of most healthy volunteers (absent in 32/34), but positive expression was observed in the blood at concentrations of 10 cells/ml or greater of the SKRC-33 RCC cell line, which is a strong expresser of cadherin-6. In peripheral blood from patients with metastatic disease, cadherin-6 mRNA was detected in 70.4% (19/27). Messenger RNA of cadherin-6 was detectable in 45.0% (27/60) of patients with localized tumors. The PCR-based detection system for peripheral blood samples from patients with metastatic disease could reveal the presence of circulating RCC cells in the blood. Detection of cadherin-6 mRNA in non-metastatic presurgical RCC patients suggests that careful follow-up study is necessary in these patients.

Radioimmunoscintigraphy of intracranial glioma xenograft with a technetium-99m-labeled mouse monoclonal antibody specifically recognizing type III mutant epidermal growth factor receptor.

The type III mutant epidermal growth factor receptor (EGFR) is expressed on the cell surface of a subset of glioma, but not of normal tissues. In this study, we investigated the in vivo kinetics of 3C10 mouse monoclonal antibody (mAb), specifically recognizing the type III mutant EGFR (EGFRvIII), using athymic nude mice bearing the intracranial glioma xenograft overexpressing the EGFRvIII. Human glioma cell line, U87MG, expressing the wild type EGFR and the transfectant, named U87MG x deltaEGFR, expressing the EGFRvIII, were transplanted subcutaneously or intracranially to nude mice. 3C10 mAb labeled with a technetium-99m (99mTc) was intravenously injected into these nude mice and then the mice were sacrificed at 24 h later, and the 99mTc-uptake by xenografts and major normal organs was measured to determine the biodistribution of mAb. Furthermore, at 3, 6 and 24 h after injection of 99mTc-labeled 3C10 mAb, whole-body scintigraphy was obtained with a gamma camera to localize the tumor site. 3C10 mAb significantly accumulated to U87MG x deltaEGFR xenografts transplanted subcutaneously or intracranially in nude mice, showing high tumor-to-blood ratio of 10.30 and 4.01, respectively. In contrast, uptake of control antibody in the intracranial tumor was as low as 0.43. In scintigrams, intracranially transplanted U87MG x deltaEGFR xenografts were detectable at 3 h after injection of 99mTc-labeled 3C10 mAb. These results suggest that intravenously injected 3C10 mAb specifically accumulated to the subcutaneous or intracranial glioma xenograft expressing the EGFRvIII and 3C10 mAb is a potential diagnostic and therapeutic agent for patients with gliomas expressing the EGFRvIII.

Induction of antigen specific cellular immunity by vaccination with peptides from MN/CA IX in renal cell carcinoma.

We investigated the induction of the specific immunity for renal cell carcinomas (RCC) using MN/CA IX, a tumor-associated antigen frequently expressed in RCC. We have generated 9-mer peptide derived from MN/CA IX and examined the antigenicity as a vaccine to induce specific immunity for RCC. To use mouse syngeneic system, we transfected human MN/CA9 cDNA into RenCa and BALB-3T3 cells originally from BALB/c mouse, and established MN/CA IX expressing mouse cell lines, i.e., MN-RenCa and MN-3T3. The immunization of BALB/c mouse with MN-RenCa cells resulted in the induction of cytotoxic T lymphocytes (CTL) against MN/CA IX expressing cells and the CTL clone was established from bulked CTL. This CTL clone specifically lyzed MN-3T3 cells, but not parental cells. To identify the targeted epitope binding to H-2Kd antigen, three 9-mer peptides (A, B, C-peptide) of human MN/CA IX compatible with the H-2Kd as well as HLA-A24 binding motif was synthesized. The cloned CTL targeted the B-peptide pulsed BALB-3T3 cells as well as MN-3T3 cells. Furthermore, spleen cells from BALB/c mouse immunized with B-peptide reacted against MN-RenCa cells. These results suggest that the peptides derived from MN/CA IX containing HLA-A24 binding motif may be useful as a potent tumor vaccine for the treatment of human RCC, and in mouse models.

Further investigation of the epitope recognized by the new monoclonal antibody 2C9.

OBJECTIVE: We established a new monoclonal antibody (2C9) that reacted with prostate tissue. The immunohistochemical reactivity of this antibody is similar to anti-prostate-specific membrane antigen (PSMA). Herein, we report the antigenic determinant of 2C9 antibody. METHODS: The reactivity of the antibody was characterized by immunohistochemical staining and the antigen target was characterized by amino acid sequencing after immuno-affinity purification from an LNCaP cell lysate and cloning of a cDNA using a mammalian expression cDNA cloning system. RESULTS: The amino acid and nucleotide sequences for the antigen molecule recognized with 2C9 monoclonal antibody demonstrated identity with PSMA. CONCLUSION: The target molecule of the 2C9 monoclonal antibody is PSMA, pointing to future diagnostic and therapeutic applications.

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles.

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.