Melanoma: Chen Y

Trefzer U, Weingart G, Chen Y, Herberth G, Adrian K, Winter H, Audring H, Guo Y, Sterry W, Walden P. · Department of Dermatology, Medical Faculty Charité, Humboldt University, Berlin, Germany. · Int J Cancer. · Pubmed #10699939 No free full text.

Abstract: Hybrid cell vaccination is a new cancer immune therapy approach that aims at recruiting T cell help for the induction of tumour specific cytolytic immunity. The vaccines are generated by fusion of the patients' tumour cells with allogeneic MHC class II bearing cells to combine the tumour's antigenicity with the immunogenicity of allogeneic MHC molecules. Safety and anti-tumour activity of this treatment were assessed in a clinical trial that has yielded one complete and one partial remission, and 5 cases of stable disease among 16 patients with advanced stage metastatic melanoma. As evidenced by histology, the vaccination induced T cell relocation into tumour nodules. Stable disease could be maintained by repeated booster injections for more than 24 months in some patients. The side effects were minor. Occasional occurrences of vitiligo spots after vaccination were indicative of a restricted therapy induced auto-immune reactivity. The results suggest that hybrid cell vaccination is a safe cancer immune therapy potentially effective for induction of acute anti-tumour response as well as long-term maintenance.

Fang J, Lu Y, Ouyang K, Wu G, Zhang H, Liu Y, Chen Y, Lin M, Wang H, Jin L, Cao R, Roque RS, Zong L, Liu J, Li T. · Institute of Chemical Industry of Forest Products, CAF, Nanjing, China. · Clin Vaccine Immunol. · Pubmed #19458203 No free full text.

Abstract: The elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer, especially in malignant melanoma of the skin, suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. We have therefore constructed a novel DNA vaccine coding for six tandem repeats of a fragment of GRP from amino acids 18 to 27 (GRP6) flanked by helper T-cell epitopes for increased immunogenicity, including HSP65, a tetanus toxoid fragment from amino acids 830 to 844 (T), pan-HLA-DR-binding epitope (PADRE) (P), and two repeats of a mycobacterial HSP70 fragment from amino acids 407 to 426 (M). The anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2) was constructed on a backbone of a pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). Intramuscular (IM) injections of anti-GRP vaccine in mice stimulated the production of high titers of specific antibodies against GRP and suppressed the growth of subcutaneous tumors of B16-F10 melanoma cells. Parallel results were obtained in vitro, showing inhibition of B16-F10 cell proliferation by GRP antisera. IM injections of the DNA vaccine also significantly attenuated tumor-induced angiogenesis associated with intradermal tumors of B16-F10 cells. In addition, lung invasion of intravenously injected cells was highly diminished, suggesting potent antimetastatic activity of the DNA vaccine. These findings support the highly immunogenic and potent antitumorigenic activity of specific anti-GRP antibodies elicited by the anti-GRP DNA vaccine.

Burns MR, Graminski GF, Weeks RS, Chen Y, O'Brien TG. · MediQuest Therapeutics, Inc, Bothell, Washington 98021, USA. · J Med Chem. · Pubmed #19281226 No free full text.

Abstract: Cancer cells can overcome the ability of polyamine biosynthesis inhibitors to completely deplete their internal polyamines by the importation of polyamines from external sources. This paper discusses the development of a group of lipophilic polyamine analogues that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. The attachment of a length-optimized C(16) lipophilic substituent to the epsilon-nitrogen atom of an earlier lead compound, D-Lys-Spm (5), has produced an analogue, D-Lys(C(16)acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375), and ovarian (SK-OV-3), among others. These results are discussed in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or nearly complete remission to this combination therapy, whereas responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response.

Chen Y, Deng W, Zhu H, Li J, Xu Y, Dai X, Jia C, Kong Q, Huang L, Liu Y, Ma C, Xiao C, Liu Y, Li Q, Bezard E, Qin C. · Department of Pathology, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 5, Panjiayuan, Nanli, Chaoyang District, Beijing, 100021, People's Republic of China. · Vet Pathol. · Pubmed #19276048 No free full text.

Abstract: Neurocutaneous melanosis (NCM) is a rare phakomatosis characterized by proliferation of melanin-producing cells in both the skin and the brain. In this study, we describe the clinical and pathologic features of NCM in a 4.5-year-old female cynomolgus macaque. Histopathologically, skin lesions showed foci of nests and cords of pigmented cells in the dermis similar to blue nevi in humans. In the brain, focal pigmented cell infiltration was observed in the connective tissue under the leptomeninges and in the brain parenchyma. The pigmented cell was moderately reactive with a pan-melanoma antibody (melanoma(pan)) in the skin. In the brain, the pigmented cell was moderately to strongly positive for melanoma(pan) in subleptomeningeal areas and in the cerebral cortex. Melanosomes were observed in pigmented cells in the brain by electron microscopic examination. Based on the histologic, immunohistochemical, and electron microscopic results, the diagnosis of NCM was made. This case is possibly the first report of the condition in animals.

Jia J, Cui J, Liu X, Han J, Yang S, Wei Y, Chen Y. · Bioinformatics and Drug Design Group, Department of Pharmacy, and Centre for Computational Science and Engineering, National University of Singapore, Singapore 117543, Singapore. · Mol Immunol. · Pubmed #19243822 No free full text.

Abstract: BACKGROUND: Tumor-specific antigens (TSAs) are potential sources of cancer vaccines, some of which are derived from T-cell epitopes of over-expressed mutant proteins to elicit immunogenicity and overcome tolerance and evasion. The lack of effective vaccines for many cancers has prompted strong interest in improved TSA search methods. Recent progresses in profiling somatic mutations and expressions of human cancer genomes, and in predicting T-cell epitopes enable genome-scale TSA search by collectively analyzing these profiles. Such a collective approach has not been explored in spite of the availability and usage of individual methods. METHODOLOGY: Genome-scale TSA search was conducted by genome-scale search of tumor-specific mutations in differentially over-expressed genes of specific cancers based on tumor-specific somatic mutation and microarray gene expression data, followed by T-cell recognition analysis of the identified mutant and over-expressed peptides to determine if they are substrates of proteasomal cleavage, TAP mediated transport and MHC-I alleles capable of eliciting immune response. The performance of our method was tested against 12 and 4 known T-cell defined melanoma and lung cancer TSAs in the Cancer Immunity database. CONCLUSIONS: Our approach identified 50% and 75% of the 12 and 4 known TSAs and predicted from the human cancer genomes additional 8-250 and 14-359 putative TSAs of 5 and 3 HLA alleles respectively. The known TSA hit rates (1.9% and 0.8%) are enriched by 29-fold and 35-fold over those of mutation analysis. The numbers of predicted TSAs are within the testing range of typical screening campaigns. Noises in expression data of small sample sizes appear to be a major factor for misidentification of known TSAs. With improved data quality and analysis methods, the collective approach is potentially useful for facilitating genome-scale TSA search.

Chen Y, Zheng W, Li Y, Zhong J, Ji J, Shen P. · State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China. · Cancer Sci. · Pubmed #19016762 No free full text.

Abstract: Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene-blue-mediated photodynamic therapy (MB-PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB-PDT under different conditions, and thereafter subjected to cell viability detection assays. MB-PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase-9/caspase-3 apoptosis pathway. Blocking activation of caspase-3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. Importantly, proteomics study defining altered protein expression in treated cells suggests the involvement of several mitochondrial proteins playing important roles in electron transfer chain, implying mitochondrial dysfunction during the treatment. Furthermore, a transplantable mouse melanoma model was utilized to estimate the effectiveness of MB-PDT in vivo. The treated mice displayed decreased tumor size and prolonged survival days, which was associated with enhanced apoptotic cell death. These results, offering solid evidence of the induction of mitochondria-related apoptosis in tumor cells, reveal new aspects of MB-PDT having potential to be a palliative treatment of melanoma.

Fan F, Feng L, He J, Wang X, Jiang X, Zhang Y, Wang Z, Chen Y. · Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China. · Carcinogenesis. · Pubmed #18515281 links to  free full text

Abstract: Raf kinase trapping to Golgi (RKTG) is a newly characterized negative regulator of the Ras-Raf-mitogen-activated and extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-signaling pathway via sequestrating Raf-1 to the Golgi apparatus. Among Raf kinase family members, B-Raf is the most frequently mutated gene in human cancers and an activated B-Raf mutation V600E is associated with >60% of human melanomas. Here, we show that RKTG can also bind and translocate B-Raf to the Golgi apparatus. When overexpressed in A375, a human malignant melanoma cell line with B-Raf(V600E), RKTG inhibits ERK activation, cell proliferation and transformation of A375 cells. In addition, the tumorigenicity of the RKTG-expressing A375 cells is suppressed in nude mice. Consistently, cell proliferation rate was reduced in the tumor xenografts in which RKTG was overexpressed. Collectively, our results suggest that RKTG may play a suppressive role in human melanoma that harbors an oncogenic B-Raf mutation via its antagonistic action on B-Raf.

Jin W, Di G, Li J, Chen Y, Li W, Wu J, Cheng T, Yao M, Shao Z. · Breast Cancer Institute, Cancer Hospital/Cancer Institute, Department of Oncology, Institute of Biomedical Science, Fudan University, Shanghai 200032, China. · FEBS Lett. · Pubmed #17659279 No free full text.

Abstract: Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.

Zhang Q, Chen Y, Wang BD, He P, Su YA. · Department of Biochemistry and Molecular Biology, the George Washington University School of Medicine and Health Sciences, Washington, DC 20037, USA. · Int J Biol Sci. · Pubmed #17657283 links to  free full text

Abstract: Introduction of human chromosome 6 into malignant melanoma cell line UACC903 resulted in generation of the chromosome 6-mediated suppressed cell subline UACC903(+6) that displays attenuated growth rate, anchorage-dependency, and reduced tumorigenicity. We have showed that overexpression of a chromosome 6-encoded tumor suppressor gene led to partial suppression to UACC903 cell growth. We now describe the differences in apoptosis and cell cycle between UACC903 and UACC903(+6) before and after UV irradiation. MTT assay revealed 86.92+/-8.24% of UACC903 cells viable, significantly (p<0.01) higher than 48.76+/-5.31% of UACC903(+6), at 24 hr after 254-nm UV irradiation (40 J/M(2)). Before UV treatment, flow cytometry analysis revealed 6.06+/-0.20% apoptosis in UACC903, significantly (p=0.01) lower than 6.67+/-0.15% in UACC903(+6). The G0/G1, S and G2/M phase cells of UACC903 were, respectively, 54.10+/-0.59%, 22.31+/-0.50% and 16.85+/-0.25%, all significantly (p<0.01) different from the corresponding percentages (58.82+/-0.35%, 20.48+/-0.05%, and 13.17+/-0.45%) of UACC903(+6). After the UV treatment, UACC903 cells in apoptosis, G0/G1, S, and G2/M became 12.59+/-0.17%, 38.90+/-0.67%, 19.74+/-0.70%, and 27.01+/-0.66%, respectively, while UACC903(+6) cells were 24.16+/-0.48%, 37.97+/-0.62%, 19.20+/-0.52%, and 15.69+/-0.14%. TUNEL assay revealed 2.31+/-0.62% apoptosis in UACC903, significantly (p<0.01) lower than 9.60+/-1.14% of UACC903(+6), and a linear and exponential increase of apoptosis, respectively, in response to the UV treatment. These results indicate that UACC903(+6) cells have a greater tendency to undergo apoptosis and are thus much more sensitive to UV irradiation. Our findings further suggest a novel mechanism for chromosome 6-mediated suppression of tumorigenesis and metastasis, i.e., through increased cell death.

Nichols LA, Chen Y, Colella TA, Bennett CL, Clausen BE, Engelhard VH. · Department of Microbiology and Carter Immunology Center, University of Virginia Health System, Charlottesville, VA 22908, USA. · J Immunol. · Pubmed #17617591 links to  free full text

Abstract: Self-tolerance to melanocyte differentiation Ags limits the ability to generate therapeutic antimelanoma responses. However, the mechanisms responsible for CD8 T cell tolerance to these Ags are unknown. We have used a newly generated TCR-transgenic mouse to establish the basis of tolerance to one such Ag from tyrosinase. Despite expression of tyrosinase transcripts in the thymus, central deletion does not shape the tyrosinase-specific CD8 T cell repertoire. We demonstrate that this endogenously expressed melanocyte Ag is constitutively presented in both peripheral and mesenteric lymph nodes, leading to abortive activation and deletion of tyrosinase-specific CD8 T cells. Importantly, this Ag is not presented by either radio-sensitive dendritic cells, or by radio-resistant Langerhans cells. Thus, for this endogenous Ag, cross-tolerization does not appear to be an operative mechanism. Instead, we find radioresistant tyrosinase mRNA expression in lymphoid compartments where CD8 T cell deletion occurs. This suggests that direct presentation of tyrosinase by radio-resistant lymph node resident cells is entirely responsible for tolerance to this endogenous melanocyte differentiation Ag.

Ganesan AK, Kho Y, Kim SC, Chen Y, Zhao Y, White MA. · Department of Dermatology, University of California, Irvine, CA 92697-2400, USA. · Proteomics. · Pubmed #17549794 No free full text.

Abstract: Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles. A proteomic SUMO target analysis in melanoma cells identified proteins involved in cellular signaling, growth control, and neural differentiation.

Benimetskaya L, Ayyanar K, Kornblum N, Castanotto D, Rossi J, Wu S, Lai J, Brown BD, Popova N, Miller P, McMicken H, Chen Y, Stein CA. · Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York, USA. · Clin Cancer Res. · Pubmed #16914583 links to  free full text

Abstract: PURPOSE: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. EXPERIMENTAL DESIGN: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. RESULTS: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors. CONCLUSIONS: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

Trefzer U, Chen Y, Herberth G, Hofmann MA, Kiecker F, Guo Y, Sterry W. · Department of Dermatology and Allergy, Skin Cancer Center, Charité - Universitätsmedizin Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany. · BMC Cancer. · Pubmed #16405722 links to  free full text

Abstract: BACKGROUND: Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested) and for metastatic melanoma of 96% (146/151 tested) in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. METHODS: In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. RESULTS: Sequence analysis of nine independent overlapping clones (length 3100-5600 bp) represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. CONCLUSION: The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

Jiang HQ, Hu XB, Li YS, Wang Y, Li YX, Wang J, Hong ZJ, Xie WG, Chen Y, Li JS. · Department of Burn and Plastic Surgery, General Hospital of Nanjing Command, Nanjing 210002, China. · Zhonghua Zheng Xing Wai Ke Za Zhi. · Pubmed #15004894 No free full text.

Abstract: OBJECTIVE: To investigate the possibility and efficacy of allograft transplantation in treating patient with huge tissue defect after radical giant malignant melanoma resection. METHODS: A male person received blood type matching was chosen as donor. Immediately after the donor's brain death, allograft was excised with the depth to the layer intervenient between periosteum and epicranial fascia in calvaria, the superficial layer of deep temporal fascia in both sides of temporal regions, close to zygomatic bones and mandibles including masseter and auricles upon in face, and cervical soft tissues including sternocleidomastoid muscles, cervical and external jugular vessels of both sides were excised simultaneously. After being perfused with 4 degrees C UW solution through both common carotid arteries, the homograft was sheared and radiated with X-ray before being preserved in UW solution for further use. During the operation, both sides of external auditory meatus were anastomosed with ears firstly, and vessels were anastomosed end-to-end sequently, at last, the border of skin flap was sutured intermittently. Combined use of MMF, FK506, Prednisone and Zenopax was performed as post-operation immunosuppressive treatment. Clinical observations were made on the signs and symptoms of graft survival or rejection as well as blood FK506 concentrations and immunological indexes were tested in laboratory. Biopsies of graft were also made at 1 h, 4 h, 8 h, 7 d, 14 d and 30 d after operation. RESULTS: The circulation of the graft was satisfactory, and the temperature and color of skin were normal. Primary healing of suture and hair growth about 0.8 cm in a month were observed. Skin Biopsies of every time had no found of hyperacute or acute rejection. The concentration of FK506 was maintained 20 mg/ml 1 month after the operation. CONCLUSION: Allograft transplantation with compound tissue of head skin flap and ears is a kind of effective and safe treatment in repairing huge tissue defect. Good tissue matching and combined use of currently available immunosuppressants can prevent hyperacute and acute rejection efficiently.

Weeraratna AT, Becker D, Carr KM, Duray PH, Rosenblatt KP, Yang S, Chen Y, Bittner M, Strausberg RL, Riggins GJ, Wagner U, Kallioniemi OP, Trent JM, Morin PJ, Meltzer PS. · Laboratory of Immunology, National Institutes of Health, The National Institute on Aging, Baltimore, MD 21224, USA. · Oncogene. · Pubmed #14755246 No free full text.

Abstract: In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

Li Y, Chen Y, Dilley J, Arroyo T, Ko D, Working P, Yu DC. · Cell Genesys, Inc., South San Francisco, CA 94080, USA. · Mol Cancer Ther. · Pubmed #14578465 links to  free full text

Abstract: Human carcinoembryonic antigen (CEA) is overexpressed in most colorectal cancers and has been widely used as a clinical marker for the management of colon cancer patients. The transcriptional regulatory elements (TREs) of CEA include two enhancer elements and a promoter in the 5'-flanking region of the CEA gene. By using these elements in different combinations to control reporter gene expression and the replication of adenovirus variants in various tumor cells, we have identified an optimal CEA regulatory cassette that tightly controls gene expression and viral replication in CEA-producing colon cancer cells. One of these variants, OV798, in which this regulatory cassette controls E1A expression, was further characterized. OV798 preferentially replicates in and kills CEA-producing colorectal cancer cell lines such as LoVo and SW1463, but its replication is attenuated by 1000-fold in the CEA-negative cell lines Colo-320DM (colon cancer), PA-1 (ovarian cancer), G361 (melanoma), U118 MG (glioma), and HBL-100 (human breast epithelial cell). The antitumor activity of OV798 was further examined in BALB/c nu/nu mice carrying s.c. human colon tumor xenografts. A single intratumoral administration of OV798 resulted in growth inhibition of human LoVo colon cancer xenografts. Six weeks after treatment, relative tumor volume decreased to 90% of baseline for the OV798 treatment group, compared to an increase to 1200% of baseline at 4 weeks for the vehicle-treated group. In vitro and in vivo characterization indicate that OV798 could be used as a therapy for human colon cancer.

Chen Y, Kramer DL, Jell J, Vujcic S, Porter CW. · Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. · Mol Pharmacol. · Pubmed #14573765 links to  free full text

Abstract: N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.

Xu XM, Chen Y, Chen J, Yang S, Gao F, Underhill CB, Creswell K, Zhang L. · Department of Oncology, Lombardi Cancer Center, Georgetown University Medical School, Washington, DC 20057, USA. · Cancer Res. · Pubmed #14522884 links to  free full text

Abstract: A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis of tumor cells. Taken together, these results suggest that the HA binding motif that is present in HA binding proteins may be responsible for the antitumor effect exerted by the members of this family.

Chen Y, Kramer DL, Li F, Porter CW. · Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. · Oncogene. · Pubmed #12902979 No free full text.

Abstract: We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.

Chen Y, Alm K, Vujcic S, Kramer DL, Kee K, Diegelman P, Porter CW. · Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. · Cancer Res. · Pubmed #12839950 links to  free full text

Abstract: The clinically relevant polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.

Chen Y, He Z, Wu L. · Department of Pathology, West China College of Stomatology, Sichuan University, Chengdu 610041, China. · Hua Xi Kou Qiang Yi Xue Za Zhi. · Pubmed #12674617 No free full text.

Abstract: OBJECTIVE: The aim of this study was to investigate the association between clinical and pathological characteristics of primary oral mucosal malignant melanoma and the prognosis of this disease. METHODS: Clinical and pathological characters of 73 cases primary oral mucosal malignant melanoma were investigated. The association between risk factors, such as black macule, lymph node metastases, invasive depth, clinic stages, pathologic types, and prognosis was analyzed using Kaplan-Meier survival analysis and Log rank test. RESULTS: The age of the patients ranged from 24 to 80 years (Median age, 50). Among the patients, 43 were males and 30 were females. The most common locations of the tumor were palate and gingiva. The clinic stages of these patients were as the following: Stage I (46/73), Stage II (24/73), Stage III (3/73). The most common pathological type was nodular (44/73), followed by lentiginose malignant melanoma (15/73) and superficial spreading (1/73). According to the configuration of tumor cells, the most common type was the mixed cell type (37/73), followed by the epitheloid cell type (27/73) and the spindle cell type (9/73). Among the 73 patients, 43 were followed up, the 3-year and 5-year survival rates were 19.86% and 11.91% respectively. Black macule, lymph node metastases, invasive depth, clinic stages, pathologic types and therapeutic methods were significantly associated with the prognosis (P < 0.05). CONCLUSION: The prognosis of the primary oral mucosal malignant melanoma is associated with black macule, lymph node metastases, invasive depth, clinic stages, pathologic types, and therapeutic methods.

Chen Y, Kamat V, Dougherty ER, Bittner ML, Meltzer PS, Trent JM. · Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Building 50, Room 5154, 50 South Drive, MSC 8000, Bethesda, MD 20892, USA. · Bioinformatics. · Pubmed #12217912 links to  free full text

Abstract: MOTIVATION: Expression-based analysis for large families of genes has recently become possible owing to the development of cDNA microarrays, which allow simultaneous measurement of transcript levels for thousands of genes. For each spot on a microarray, signals in two channels must be extracted from their backgrounds. This requires algorithms to extract signals arising from tagged mRNA hybridized to arrayed cDNA locations and algorithms to determine the significance of signal ratios. RESULTS: This paper focuses on estimation of signal ratios from the two channels, and the significance of those ratios. The key issue is the determination of whether a ratio is significantly high or low in order to conclude whether the gene is upregulated or downregulated. The paper builds on an earlier study that involved a hypothesis test based on a ratio statistic under the supposition that the measured fluorescent intensities subsequent to image processing can be assumed to reflect the signal intensities. Here, a refined hypothesis test is considered in which the measured intensities forming the ratio are assumed to be combinations of signal and background. The new method involves a signal-to-noise ratio, and for a high signal-to-noise ratio the new test reduces (with close approximation) to the original test. The effect of low signal-to-noise ratio on the ratio statistics constitutes the main theme of the paper. Finally, and in this vein, a quality metric is formulated for spots. This measure can be used to decide whether or not a spot ratio should be deleted, or to adjust various measurements to reflect confidence in the quality of the measurement. CONTACT: e-dougherty@tamu.edu

Zhang J, Ramesh N, Chen Y, Li Y, Dilley J, Working P, Yu DC. · Cell Genesys, Inc., Foster City, California 94404, USA. · Cancer Res. · Pubmed #12097284 links to  free full text

Abstract: Uroplakins (UPs) are a group of integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but very little is known about its transcription response elements. To identify the promoter elements, a DNA fragment of 2239 bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed that the DNA segment located between -1809 and +1 bp resulted in preferential expression in bladder carcinoma cells with negligible expression in nonurothelial cells. This promoter was engineered into adenovirus (Ad) type 5 to drive the expression of the E1A and E1B genes and to create an attenuated replication-competent Ad variant, termed CG8840. Viral replication and the cytopathic effect of CG8840 were evaluated by virus yield and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in bladder transitional cell carcinoma (TCC) cell lines RT4 and SW780; nonbladder cancer cell lines G361 (melanoma), LNCaP (prostate cancer), PA-1 (ovarian cancer), and U118 (brain cancer); and human primary cells including lung fibroblasts, bladder smooth muscle cells, and mammary epithelial cells. CG8840 replicated in and eliminated bladder TCC efficiently with high specificity (10,000:1) in comparison with nonbladder cells. The antitumor activity of CG8840 was examined in BALB/c nu/nu mice carrying s.c. human TCC xenografts. Intratumoral and i.v. administration of CG8840 in RT4 human bladder cancer xenografts caused significant (P < 0.01) inhibition of tumor growth. Synergistic antitumor efficacy was observed when CG8840 was combined with docetaxel, resulting in significant regression of RT4 bladder cancer xenograft tumors within 6 weeks after i.v. administration of CG8840 (3.33 x 10(9) plaque-forming units/animal on day 1) and docetaxel (20 mg/kg on days 2, 6, and 9). These results demonstrate the utility of the UPII promoter in the generation of urothelium-specific adenoviral vectors and provide a potential foundation for the development of bladder tumor-specific oncolytic viral therapies.

Li S, Chen Y, Wei H. · Institute of Pathology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. · Zhonghua Bing Li Xue Za Zhi. · Pubmed #11758218 No free full text.

Abstract: OBJECTIVE: Random amplified polymorphic DNA (RAPD) analysis was used to detect the genomic variant in B16 melanoma, and to seek the tumor related DNA fragments. METHODS: Genomic DNA from B16 melanoma and C57BL/6J mouse normal tissues were amplified by RAPD with 105 random primers, the significantly different DNA fragments were isolated, cloned and sequenced. DNA sequences were analyzed with GenBank data. RESULTS: 24 of the 105 primers generated polymorphic profiles when the B16 melanoma RAPD profile was compared to that of its normal tissue DNA. By amplification of the primer AB8-5, a tumor band showing loss with respect to normal band was detected and this DNA fragment was designated as B8-5. B8-5 was a 610 bp fragment, the sequencing result of B8-5 showed 99% homology with muscle pigment epithelium-derived factor (PEDF) gene, and 100% homology with muscle pigment epithelium-derived factor mRNA. B8-5 was therefore regarded as PEDF gene. CONCLUSION: Our result found that allelic losses exist in PEDF gene, and therefore suggest that PEDF is associated with tumorigenesis of B16 melanoma.

Dan Q, Sanchez R, Delgado C, Wepsic HT, Morgan K, Chen Y, Jeffes EW, Lowell CA, Morgan TR, Jadus MR. · Diagnostic and Molecular Medicine Health Care Group, Veterans Affairs Medical Center, University of California, 5901 E. 7th Street, Long Beach, CA 90822, USA. · Mol Ther. · Pubmed #11708879 links to  free full text

Abstract: Hepatocellular carcinoma is a lethal disease and methods that develop effective cellular-based immunotherapy are needed. We retrovirally transduced non-immunogenic mouse Hepa1-6 hepatoma cells with the gene encoding the membrane form of macrophage colony stimulating factor (mM-CSF). Excess recombinant M-CSF and phagocytosis-inhibiting chemicals blocked macrophage-mediated killing of cloned mM-CSF transfected Hepa1-6 hepatoma cells. Macrophages derived from Hck(-/-)Fgr(-/-) and Lyn(-/-) triple knockout mice, which are incapable of performing phagocytosis, failed to kill the mM-CSF transduced cells. The mM-CSF transfected tumor clones failed to grow when injected into C57BL/6 or C57L/J mice. Splenocytes from these vaccinated mice displayed cytotoxicity against parental Hepa1-6 cells, but not against B16 and CT-26 tumor cells in vitro. Mice that rejected the mM-CSF transfected Hepa1-6 tumor subsequently rejected parental Hepa1-6 cells but not the B16 melanoma cells when rechallenged. Elimination of the CD8+ effector cells by an anti-CD8 antibody and complement treatment prevented the adoptive transfer of anti-Hepa1-6-specific immunity into naive animals. Thus, mM-CSF provides a method of generating effective anti-tumor immune responses by macrophages and cytotoxic T cells against the parental Hepa1-6 cells. Our work suggests that mM-CSF transduced hepatoma cells could be used as a tumor vaccine to stimulate immune responses against hepatocellular carcinoma.