Prognostic impact and immunogenicity of a novel osteosarcoma antigen, papillomavirus binding factor, in patients with osteosarcoma

To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a cytotoxic T lymphocytes (CTL)-defined osteosarcoma antigen in the context of human leukocyte antigen (HLA)-B55. In the present study, we analyzed the distribution profile of PBF in 83 biopsy specimens of osteosarcomas and also the prognostic impact of PBF expression in 78 patients with osteosarcoma who had completed the standard treatment protocols. Next, we determined the antigenic peptides from PBF that react with peripheral T lymphocytes of HLA-A24⁺ patients with osteosarcoma. Immunohistochemical analysis revealed that 92% of biopsy specimens of osteosarcoma expressed PBF. PBF-positive osteosarcoma conferred significantly poorer prognosis than those with negative expression of PBF ( P =  0.025). In accordance with the Bioinformatics and Molecular Analysis Section score, we synthesized 10 peptides from the PBF sequence. Subsequent screening with an HLA class I stabilization assay revealed that peptide PBF A24.2 had the highest affinity to HLA-A24. CD8⁺ T cells reacting with a PBF A24.2 peptide were detected in eight of nine HLA-A24-positive patients with osteosarcoma at the frequency from 5 × 10⁻⁷ to 7 × 10⁻⁶ using limiting dilution/mixed lymphocyte peptide culture followed by tetramer-based frequency analysis. PBF A24.2 peptide induced CTL lines from an HLA-A24-positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA-A24. These findings suggested prognostic significance and immunodominancy of PBF in patients with osteosarcoma. PBF is the candidate target for immunotherapy in patients with osteosarcoma. ( Cancer Sci 2008; 99: 368–375)     

Identification of novel human leukocyte antigen‐A*11:01‐restricted cytotoxic T‐lymphocyte epitopes derived from osteosarcoma antigen papillomavirus binding factor

Osteosarcoma is the most common malignancy of bone that affects young people. Neoadjuvant chemotherapy and surgery have significantly improved the prognosis. However, the prognosis of non‐responders to chemotherapy is still poor. To develop peptide‐based immunotherapy for osteosarcoma, we previously identified CTL epitopes derived from papillomavirus binding factor (PBF) in the context of human leukocyte antigen (HLA)‐A2, HLA‐A24 and HLA‐B55. In the present study, we identified two novel CTL epitopes, QVT (QVTVWLLEQK) and LSA (LSALPPPLHK), in the context of HLA‐A11 using a sequence of screenings based on the predicted affinity of peptides, in vitro folding ability of peptide/HLA‐A11 complex, reactivity of peptide/HLA‐A11 tetramer and interferon (IFN)‐γ production of T cells that was induced by mixed lymphocyte peptide culture under a limiting dilution condition. CTL clones directed to QVT and LSA peptides showed specific cytotoxicity against HLA‐A11⁺PBF⁺ osteosarcoma (HOS‐A11) cells. In contrast, another epitope, ASV (ASVLSRRLGK), could highly induce cognate tetramer‐positive CTL. This might be because the ASV peptide mimics the peptide ASV (R6Q) (ASVLSQ RLGK) derived from bacterial polypeptides, ROK family proteins. However, ASV‐induced CTL did not show cytokine production against the cognate peptide. In conclusion, the CTL epitopes QVT and LSA peptides might be useful for the development of immunotherapy targeting PBF for patients with osteosarcoma.     

Identification of human papillomavirus DNA gene sequences in human breast cancer

Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at -70 degrees C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast.     

Identification by monoclonal antibody of the tumor antigen of a human autologous breast cancer cell that is involved in cytotoxicity by a cytotoxic T-cell clone

We have already established a pair of human autologous clones, tumor-specific cytotoxic T-lymphocyte clone TcHMC-1 and tumor target clone HMC-1-8, that were derived from the metastatic pleural effusion of a patient with mammary carcinoma. In this paper, we describe the target antigen that was defined by monoclonal antibody 3A2. This monoclonal antibody selectively inhibited the cytotoxic action of TcHMC-1 against HMC1-8 autologous tumor target cells, but not the cytotoxicity of lymphokine-activated killer and possibly natural killer cells against HMC-1-8 cells. Western blot analysis using the 3A2 monoclonal antibody identified a molecule with an approximate molecular weight of 92,000. This antigen was highly expressed on autologous primary cancer cells of breast carcinoma tissue, but not on the normal mammary gland in the same patient. Moreover, this antigen can be detected on approximately 50% of human allogeneic breast carcinomas, but not on other neoplastic tissues such as gastric and colonic carcinomas except for one out of 10 prostatic carcinomas. Nonneoplastic normal cells did not express this antigen. It was also suggested that the antigen is not murine mammary tumor virus-related products. These data suggest that 3A2-defined antigen could participate in the cytotoxicity by human autologous cytotoxic T-lymphocytes as the target molecule expressed on tumor cells.     

Identification of a human glioma-associated growth factor gene, granulin, using differential immuno-absorption

Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3-30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early-passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms.     

Identification and characterization of a p53 gene mutation in a human osteosarcoma cell line

The nuclear phosphoprotein p53 occurs at elevated levels in many transformed cells. Mutant forms of mouse p53 possess enhanced transforming activity compared with wild type p53. Mutant mouse p53 proteins form complexes with the 70 kDa family of heat shock proteins (HSPs). We previously demonstrated an association between p53 and the 70 kDa HSPs in the human osteosarcoma (HOS) derived cell line HOS-SL. We report here the molecular cloning and sequencing of the p53 gene from HOS-SL cells, and demonstrate that it is in fact mutant. Further, analysis of similar HOS-derived cell lines demonstrates that they also encode the same mutant form of p53, whereas the wild type form of p53 appears to be lost in these cells. Stability studies demonstrate an increased half life of the p53 protein in these cells, in keeping with its association with the HSP 70 proteins. A potential role for this p53 mutant in the transformation process is discussed.     

Augmentation of human cytotoxic T lymphocytes against autologous tumor by a factor released from human monocytic leukemia cell line

A human acute monocytic leukemia cell line, THP-1, releases a factor which activates human cytotoxic (killer) T lymphocytes (CTL) against autologous tumor in vitro. The factor, named cytotoxic (killer) T cell activating factor (KAF), is an acidic protein of 70,000 to 100,000 dalton molecular size. Peripheral blood leukocytes from two patients, bearing epithelioid sarcoma or malignant schwannoma, were cultured for 7 days with individual autologous tumor to induce CTL directed to the corresponding tumor. Monocyte-depleted peripheral leukocytes generated lesser CTL activity than the monocyte-containing leukocyte population. However, the KAF was able to replace the monocyte function. The KAF acted at the CTL generation phase as well as the effector phase. The KAF-activated killer cells possessed CD4-8+ surface phenotype. The CTL killed autologous tumor or other unrelated tumor cell lines only when they shared some of the HLA class I antigens. It was also demonstrated that the KAF does not activate killer cells without proper antigenic stimuli, because the KAF-augmented CTL possess specificity against autologous tumor or other HLA-A or -B matched tumor cell lines. The therapeutic applicability of human KAF for anti-tumor CTL therapy against autologous tumor is discussed.     

A rearranged EP300 gene in the human B-cell lymphoma cell line RC-K8 encodes a disabled transcriptional co-activator that contributes to cell growth and oncogenicity

Previously, we reported that a novel secretory protein, pancreatic adenocarcinoma up-regulated factor (PAUF), which is highly expressed in pancreatic cancer and mediates the growth and metastasis of pancreatic cancer cells. In this study, we generated and characterized a 2′-fluoropyrimidine modified RNA aptamer (P12FR2) directed against human PAUF. P12FR2 binds specifically to human PAUF with an estimated apparent K_D of 77 nM. P12FR2 aptamer inhibits PAUF-induced migration of PANC-1, human pancreatic cancer cells, in a wound healing assay. Moreover, intraperitoneal injection of P12FR2 decreased tumor growth by about 60% in an in vivo xenograft model with CFPAC-1 pancreatic cancer cells, without causing a loss of weight in the treated mice. Taken together, we propose here that PAUF-specific RNA aptamer, P12FR2, has the potential to be effective in the therapy of human pancreatic cancer.     

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.     

Identification of a human T cell clone with the cytotoxic T lymphocyte and natural killer-like cytotoxic function against autologous mammary carcinoma and K562 line

Pleural exudative lymphocytes (PLEL) from a 60-year-old female patient showed high cytotoxicity against the autologous mammary tumor line, HMC-2, and NK-susceptible K562 cells, although PLEL demonstrated only weak cytotoxic potentials against several allogeneic tumor lines. We successfully obtained seven cytotoxic T cell clones from PLEL bulk populations, and assessed the possibility that these lymphocytes are simply natural killer (NK)-like cells or have the dual cytotoxic activity of cytotoxic T lymphocytes (CTL) and NK-like cells. These clones, designated as TcHMC-2, showed strong cytotoxicity against both HMC-2 and K562 cells. In contrast, allogeneic human peripheral blood-derived NK cells could not kill HMC-2 targets. Furthermore, a blocking study of TcHMC-2 cytotoxicity using monoclonal antibodies against CD3, CD8 and human MHC class I products showed that all of these antigen molecules were involved in the cytotoxicity of TcHMC-2 clone against autologous HMC-2 cells, indicating MHC class I recognitive cytotoxicity. These data indicate that the TcHMC-2 clone may have dual cytotoxicity with CTL- and NK-like activity against autologous HMC-2 mammary tumor and K562 cells, respectively.     

拮抗凋亡转录因子基因在高转移人成骨肉瘤细胞系中的高表达

背景与目的：成骨肉瘤是青少年中最常见的恶性骨肿瘤,具有易发生肺转移的特点。尽管目前已能够成功地控制原发瘤,但是仍有30％以上的患者在5年内死于肺转移。更好地理解调节转移过程的分子机制,为设计有效的治疗方案提供生物学基础,我们用原位移植的方法建立了人成骨肉瘤细胞系SOSP－9607裸鼠转移模型,用这一模型筛选出了高转移细胞系SOSP－M。本研究旨在通过比较两个细胞系的基因表达水平来克隆与骨肉瘤转移相关的基因,探讨成骨肉瘤转移的分子机制。方法：采用抑制性消减杂交技术建立人成骨肉瘤高转移细胞株SOSP－M的消减文库;用差异筛选技术筛选出阳性克隆;对部分克隆进行测序和同源性分析,用Northern杂交后转录多聚酶链式反应技术对SOSP－M中表达上调的有意义的克隆在低转移细胞系SOSP－9607,OS－9901,高转移细胞系SOSP－M和裸鼠肺转移结节中的表达情况进行了分析。结果：在高转移细胞株SOSP－M中有2个阳性克隆均与人凋亡对抗转录因子（apoptosis antagonizing transciption factor)同源（99％同源）。Northern杂交及反转录PCR证实这个基因在高转移细胞和裸鼠肺转移结节中高表达,而在低转移人成骨肉瘤细胞系SOSP－9607和OS－9901中表达微弱。结论：对抗凋亡转录因子在促进肿瘤细胞转移中可能起重要作用。     

Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.     

Identification of RB1CC1, a novel human gene that can induce RB1 in various human cells

Multidrug resistance to anti-cancer agents (MDR) is a major barrier to successful cancer treatment. Current knowledge about genes that contribute to MDR is limited, however, and its mechanisms remain unclear. To identify genes involved in MDR, we performed differential display analysis and isolated a novel human gene, RB1CC1 (RBI-inducible Coiled-Coil 1). The 6.6-kb RB1CC1 cDNA encodes a putative 1594-amino-acid protein that contains a nuclear localization signal, a leucine zipper motif and a coiled-coil structure. Western blot analysis and immunocytochemical staining with anti-RB1CC1 antibody showed that endogenously expressed RB1CC1 protein localized to the nucleus. In MDR variants of human osteosarcoma cells, RB1CC1 expression increased in response to doxorubicin-induced cytotoxic stress and remained elevated for the duration of drug treatment. RB1CC1 expression levels correlated closely with those of RB1 (retinoblastoma 1) in cancer cell lines as well as in various normal human tissues. Moreover, introduction of wild-type RB1CC1 significantly induced RB1 expression in human leukemic cells. These data suggest that RB1CC1 may be a key regulator of RB1 gene expression.