A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer.

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3'-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

Nuclear ß-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer.

BACKGROUND: Although the prognostic roles of ß-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein. METHOD: In this study, we have evaluated the expression levels and subcellular localization of ß-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome. RESULTS: We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear ß-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear ß-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of ß-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear ß-catenin rather than cytoplasmic ß-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of ß-catenin can not be obviously enhanced by adding EGF. CONCLUSION: We propose that evaluation of subcellular localization of ß-catenin and Nanog expression is of clinical significance for patients with NSCLC.

[A murine model of human primary hepatic lymphoma constructed by surgical orthotopic implantation of histologically intact human tumor tissue and in vivo continuous orthotopic passage].

OBJECTIVE: To establish a nude murine model of human primary hepatic lymphoma (PHL) with surgical orthotopic implantation of histologically intact human tumor tissue and in vivo continuous orthotopic passage. METHODS: Histologically intact lymphoma tissues harvested intraoperatively from a PHL patient were orthotopically transplanted into liver parenchyma of nude mice and in vivo continuous orthotopic passage in nude mice was used to develop a nude murine model mimicking the biological characteristics of PHL patients. Histopathology (light microscopy and immunohistochemistry), serological test, karyotypic analysis and flow cytometry were used to explore the tumorigenicity, invasion and metastasis. RESULTS: An orthotopic nude murine model of PHL, named HLBL-0102, was successfully developed. Histopathology of transplanted tumors showed primary hepatic lymphoma (diffuse large B cell) stained positive for CD20, CD79a and MUM1. Serological test in tumor-bearing mice indicated that alpha-fetal protein (AFP) was negative and hepatitis B surface antigen (HBsAg) positive. The serum level of lactate dehydrogenase (LDH) was elevated to an average of ((1223 ± 258) vs (124 ± 54) U/L, P < 0.01). The chromosomal number of transplanted tumors was between 55 and 59. The DNA index (DI) of 1.7 ± 0.2 indicated heteroploid. So far HLBL-0102 model has been passed for 42 generations in nude mice. A total of 320 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumors were both 100%. The transplanted tumors grew invasively in the liver of nude mice and destroyed adjacent liver tissues and bile ducts, veins and arteries of portal area. There was no involvement of other tissues, organs and distal lymph nodes. CONCLUSION: An orthotopically transplanted model has been successfully established for human primary hepatic lymphoma in nude mice.

A liver-metastatic model of human primary gastric lymphoma in nude mice orthotopically constructed by using histologically intact patient specimens.

BACKGROUND AND OBJECTIVE: In recent years, incidence and mortality of lymphoma are markedly increasing worldwide. However, the pathogenesis and mechanism of invasion and metastasis for lymphoma are not yet fully clarified. It is mainly due to the lack of ideal animal models, which can precisely simulate the invasion and metastasis of lymphoma in the human body. So, it is very necessary to establish a highly metastatic nude mouse model of human lymphoma. This study developed a liver-metastatic model of primary gastric lymphoma in nude mice by using orthotopic surgical implantation of histologically intact patient specimens into the corresponding organs of the recipient small animals. METHODS: A histologically intact fragment of liver metastasis derived from a surgical specimen of a patient with primary gastric lymphoma was implanted into the submucosa of the stomach in nude mice. Tumorigenicity, invasion, metastasis, morphologic characteristics (via light microscopy, electron microscopy, and immunohistochemistry), karyotype analysis, and DNA content of the orthotopically transplanted tumors were studied. RESULTS: An orthotopic liver metastatic model of human primary gastric lymphoma in nude mice (termed HGBL-0304) was successfully established. The histopathology of the transplanted tumors showed primary gastric diffuse large B-cell lymphoma. CD19, CD20, CD22, and CD79alpha were positive, but CD3 and CD7 were negative. The serum level of lactate dehydrogenase (LDH) was elevated [(1010.56+/-200.85) U/L]. The number of chromosomes ranged from 75 to 89. The DNA index (DI) was 1.45+/-0.25 (that is, heteroploid). So far, the HGBL-0304 model has been passed on for 45 generations of nude mice. A total of 263 nude mice were used for the transplantation. Both the growth and resuscitation rates of liquid nitrogen cryopreservation of the transplanted tumors were 100%. The transplanted tumors autonomically invasively grew and damaged a whole layer in the stomach of nude mice. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 100%, 94.3%, 62.6%, and 43.5%, respectively. CONCLUSIONS: The study successfully establishes an orthotopic liver metastatic model of human primary gastric lymphoma in nude mice. The HGBL-0304 model can completely simulate the natural clinical process of primary gastric lymphoma and provides an ideal animal model for the research on the biology of metastasis and antimetastatic experimental therapies of primary gastric lymphoma.

[Highly metastatic nude mouse model of human primary gastric lymphoma constructed by surgical orthotopic transplantation and in vivo continuous screening method].

OBJECTIVE: To develop a series of high metastatic models of human gastric malignant lymphoma in nude mice by orthotopic transplantation. METHODS: Two histologically intact primary and hepatic metastatic fragments derived from surgical specimen of a patient with primary gastric lymphoma were implanted into the submucosa of stomach in nude mice. Highly metastatic and specific organ metastatic models were screened by selective orthotopic passage in nude mice. Transplantability, invasion, metastasis, morphological characteristics (light microscopy, electron microscopy and immunohistochemistry), karyotypic analysis and DNA content of orthotopically transplanted tumors were studied. RESULTS: Primary and hepatic metastatic fragments of primary gastric lymphoma were successfully transplanted in nude mice. Two nude mouse models of human primary gastric lymphoma, termed HGBL-0304 (hepatic metastasis model) and HGBL-0305 (high metastasis model), were developed, exhibiting different metastasis biology. Histopathology of transplanted tumors showed primary gastric diffuse large B cell lymphoma. Two models have been maintained for 45 generations by orthotopic passage in nude mice. A total of 419 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumors were both 100%. Significant difference in metastasis biology was exhibited in four aspects of metastasis time, organ metastatic rate, the extent of hepatic metastasis and survival of cancer-bearing mice. The metastatic rates of liver, spleen, lymph nodes and peritoneal seeding in HGBL-0304 and HGBL-0305 models were 100% and 69.5%, 94.3% and 55.6%, 62.6% and 45.7%, and 43.5% and 30.5%. The onset time for metastases of liver, spleen, lymph nodes and peritoneal seeding was 2 w and 5 w, 3 w and 6 w, 2 w and 3 w, 3 w and 6 w respectively. The extent of hepatic metastasis in HGBL-0304 and HGBL-0305 models displayed diffuse involvement of the whole liver and mainly right lobe invasion of liver respectively. The mean survival time of HGBL-0304 and HGBL-0305 models was 54.3d and 106.9 d respectively. CONCLUSION: Surgical orthotopic implantation combined with in vivo selective passage screening is an effective method for establishing highly metastatic and specific organ metastatic models of human malignant lymphoma in nude mice. The study is the first time to establish hepatic metastasis and high metastasis nude mouse models of human primary gastric lymphoma with the same original patient and different potentials of invasion and metastasis.

[Establishment of a highly-metastatic model of human primary melanoma of the small intestine orthotopically transplanted in the small intestine of nude mice].

OBJECTIVE: To provide an useful animal model for exploring metastatic biology and anti-metastatic therapy of primary malignant melanoma of the small intestine. METHODS: A 49-year old male patient with malignant melanoma was treated by surgery, and the primary tumor in the small intestine and a metastatic tumor in the liver were removed. The diagnosis of malignant melanoma was confirmed by histopathology. Fresh melanoma tissue fragments taken from the primary intestinal tumor and hepatic metastatic tumor were orthotopically implanted into the mucosal layer of small intestine in nude mice, respectively. The tumor growth rate, invasion and metastasis of the transplanted tumors were observed. Light and electron microscopy, immunophenotype analysis, flow cytometry and karyotype analysis were carried out. RESULTS: Fragments of the primary and liver metastatic malignant melanoma were successfully implanted in nude mice. After continuous passages in nude mice, an highly-metastatic model of human primary malignant melanoma of the small intestine (from the primary lesion) in nude mice (termed HSIM-0602) and a liver metastatic model of human primary malignant melanoma of the small intestine (originally from the liver metastatic lesion) in nude mice (termed HSIM-0603) were successfully established. Histological examination of the transplanted tumors revealed a high-grade melanoma of the small intestine. Immunohistochemical stainings of S-100 protein and HMB45 were positive. Many scattered melanosomes and melanin complex were seen in the cytoplasm of tumor cells. Chromosomal modal number was between 55 and 59. DNA index (DI) was 1.59 - 1.71, representing a heteroploid. The HSIM-0602 and HSIM-0603 tumor models had been maintained for 21 and 23 passages in nude mice, respectively. 227 nude mice were used for transplantation. Both the growth rate after transplantation and resuscitation rate from liquid nitrogen cryopreservation were 100%. The HSIM-0602 model exhibited 84.8% lung metastasis, 65.7% liver metastasis and 63.8% lymph node metastasis. However, HSIM-0603 displayed 100% liver metastasis, 46.7% lung metastasis and 71.3% lymph node metastasis. The transplanted tumors actively and invasively grew in the small intestine of nude mice and showed hematogenous and lymphatic metastases. CONCLUSION: To our knowledge it is the first time that two strains of spontaneous highly-metastatic nude-mouse model of human primary malignant melanoma of the small intestine have been successfully established in our department. The models are very closely mimic the natural clinicopathologic course of primary small intestinal melanoma in humans and provide ideal animal models for the researches on metastasis biology and anti-metastatic experimental therapy of malignant melanoma of the small intestine.

Sulforaphane inhibits cancer stem-like cell properties and cisplatin resistance through miR-214-mediated downregulation of c-MYC in non-small cell lung cancer.

We herein report that sulforaphane (SFN), a potent anti-cancer and well-tolerated dietary compound, inhibits cancer stem-like cell (CSC) properties and enhances therapeutic efficacy of cisplatin in human non-small cell lung cancer (NSCLC). SFN exerted these functions through upregulation of miR-214, which in turn targets the coding region of c-MYC. This finding was further corroborated by our observations that plasmid or lentiviral vector-mediated expression of 3'UTR-less c-MYC cDNA and cisplatin- or doxorubicin-induced endogenous c-MYC accumulation was similarly suppressed by either SFN or miR-214. Further, we showed that the reported inhibitory effects of SFN on ß-catenin are also mediated by miR-214. SFN/miR-214 signaling inhibited CSC properties and enhanced the cytotoxicity of chemotherapeutic drugs in vitro. Experiments with nude mice carrying xenograft tumors showed that SFN sensitized NSCLC cells to cisplatin's efficacy, which is accompanied by inhibition of cisplatin-induced c-MYC accumulation in tumor tissues. Our results provided strong evidence and mechanisms to support consideration of SFN or synthetic derivatives as a therapeutic agent in combination with cisplatin for the treatment of patients with NSCLC and, potentially, other types of c-MYC-addicted tumors.

[Establishment of a nude mouse model of orthotopically transplanted human primary malignant lymphoma of the liver].

OBJECTIVE: To establish a nude mouse model of orthotopically transplanted human primary malignant lymphoma of the liver, and to serve researches on pathogenesis and experimental treatment of this disease. METHODS: Small pieces of lymphoma tissues freshly taken from patients with primary lymphoma of the liver were orthotopically transplanted into the liver parenchyma in nude mice. Tumorgenicity, invasion, metastasis, and morphological characteristics were examined by light and electron microscopy and immunohistochemistry. AFP, HBsAg and LDH were assayed by serological test. Karyotype analysis and DNA content of orthotopically transplanted tumors were also performed. RESULTS: A nude mouse model of orthotopically transplanted human primary malignant lymphoma of the liver was successfully established and named HLBL-0102. The tumor was confirmed as primary lymphoma of the liver (non-Hodgkin's lymphoma, B cell) by histopathology. Immunohistochemistry showed positive expression of CD19, CD20, CD45 and CD79a, but negative of CD3 and CD7. Serological test indicated that AFP was negative, HBsAg positive and the concentration of LDH was 1267.5 U/L. The number of chromosomes was between 55 and 59. DNA index (DI) was 1.57 approximately 1.61 (i.e. heteroploid). So far, the strain HLBL-0102 has grown for 3 years and been passaged for 37 generations in nude mice. Totally 283 nude mice were used for transplantation and the successful rate was 100%. Both the growth rate and resuscitation rate of liquid nitrogen cryo-preserved transplanted tumors were 100%. The transplanted tumors grew intensely and invasively in the liver of nude mice and damaged adjacent liver tissues, bile ducts and portal vein areas. No involvement of other tissues and organs and distal lymph nodes was observed. CONCLUSION: To our knowledge it is the first report of successfully established nude mouse model of orthotopically transplanted human primary malignant lymphoma of the liver. The HLBL-0102 model simulate very well the natural process of human primary lymphoma of the liver and provides an ideal animal model for researches on the biology and therapies of this malignancy.

Effects of different concentrations of amino acids in the culture medium on preimplantation mouse embryo development in vitro.

OBJECTIVE: To evaluate the effects of amino acids (AA) on the development of in vitro cultured preimplantation embryos of Kunming mice, and define the optimal AA concentration for embryo culture. METHODS: Totally 630 zygotes were collected from the oviducts of superovulated female Kunming mice, which were cultured in protein-free potassium simplex optimized medium (mKSOM) supplemented with Eagle's essential amino acids and Eagle's non-essential amino acids of different concentrations (mKSOM, mKSOM+1/16AA, mKSOM+1/8AA, mKSOM+1/4AA, mKSOM+1/2AA, mKSOM+AA, and mKSOM+2AA). RESULTS: The embryos cultured with the amino acids showed higher development rate to both 8-cell embryo stage and blastocyst stage than those cultured without amino acids. The correlation of amino acid concentration with 8-cell and blastocyst development rates conformed to the cubic model, with the highest development rate to both of the two stages observed at half of the amino acid concentration. CONCLUSION: Amino acids can promote the development of preimplantation Kunming mouse embryos, but excessively high concentration of amino acids impair embryo development possibly because of metabolic and osmotic pressure changes of the embryos as well as toxicity of ammonium resulting from the metabolism of amino acids.

[Establishment and characteristics of orthotopically transplanted model of human primary malignant small intestinal lymphoma in nude mice].

OBJECTIVE: To establish orthotopically transplanted model of human malignant small intestinal lymphoma in nude mice and analyze their biologic characteristics. METHODS: Small intestinal lymphoma tissues from 5 patients were transplanted into intestinal mucosa of nude mice. Tumorgenecity, invasion and metastasis of the transplanted tumors were observed by morphological analyses (light microscopy, electron microscopy and immunohistochemistry), karyotyping and DNA quantitative assay. RESULTS: Tumor tissues from 3 lymphoma patients were successfully transplanted. According to the World Health Organization classification, the three models were classified into non-Hodgkin's lymphoma (B cell) of human small intestine (HSIL-1), high metastasis of non-Hodgkin's lymphoma (B cell) of human small intestine (HSIL-2) and non-Hodgkin's lymphoma (T cell) of human small intestine (HSIL-3), respectively. Immunohistochemistry showed that CD19, CD20, CD22, CD40, CD45 and CD72 were positive in HSIL-1 and HSIL-2, whereas CD3, CD7 and CD45RO were positive in HSIL-3. The karyotypes of the transplanted tumors were all hypotriploid with modal numbers from 55 to 69 and the DNA index (DI) was 1.46 approximately 1.71. The three models had been passaged for 32, 27 and 21 generations respectively in 433 nude mice. The growth rate, resuscitation rate of the liquid nitrogen preserved tumor cells and spontaneous metastasis rate upon transplantation were all 100%. We observed an invasive growth of the transplanted tumors in small intestine, which resulted in disrupting of the intestinal wall, hematogenous metastasis, lymph node metastasis and seeding metastasis. The features of the transplanted tumors were similar to the original tumors in histopathology, ultrastructure, DNA content and karyotype. CONCLUSION: Three strains of orthotopically transplanted model of human primary malignant small intestinal lymphoma in nude mice were successfully developed. The result of research will provide ideal animal models for further studies on mechanism of tumorigenesis, invasion and metastasis of malignant small intestinal lymphoma and experimental therapy.

Cancer stem-like cell properties are regulated by EGFR/AKT/ß-catenin signaling and preferentially inhibited by gefitinib in nasopharyngeal carcinoma.

We report that the epidermal growth factor receptor (EGFR) pathway plays a critical role in regulating cancer stem-like cells (CSCs) in nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia. Effects of EGFR on maintaining CSCs are mainly mediated by AKT signaling, and ß-catenin is responsible for governing CSC properties in response to EGFR/AKT activation. Significantly, CSCs are enriched by cisplatin and decreased by gefitinib in NPC xenograft models. Upon reimplantation in secondary mice, tumor cells derived from cisplatin-treated mice grew rapidly, whereas regrowth of tumor cells from gefitinib-treated mice was severely diminished. We further demonstrate that expression of EGFR correlates with expression of ß-catenin and Nanog in primary tumor specimens from NPC patients. These findings provide mechanistic and preclinical evidence supporting the use of gefitinib alone or in combination with a chemotherapeutic agent in first-line therapy for patients with NPC. In addition, our results suggest that targeting ß-catenin represents a rational clinical modality for patients whose tumors harbor activated EGFR or AKT.

Antisense oligonucleotide against hTERT (Cantide) inhibits tumor growth in an orthotopic primary hepatic lymphoma mouse model.

BACKGROUND: Human xenograft models, resulting from orthotopic transplantation (implantation into the anatomically correct site) of histologically intact tissue into animals, are important for investigating local tumor growth, vascular and lymphatic invasion at the primary tumor site and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We used surgical orthotopic transplantation to establish a nude mouse model of primary hepatic lymphoma (PHL), HLBL-0102. We performed orthotopic transfer of the HLBL-0102 tumor for 42 generations and characterized the tumor cells. The maintenance of PHL characteristics were supported by immunohistochemical and cytogenetic analysis. We also report the antitumor effect of Cantide, an antisense phosphorothioate oligonucleotide against hTERT, on the growth of HLBL-0102 tumors. We showed a significant, dose-dependent inhibition of tumor weight and serum LDH activity in the orthotopically transplanted animals by Cantide. Importantly, survival was prolonged in Cantide-treated HLBL-0102 tumor-bearing mice when compared to mock-treated mice. CONCLUSIONS/SIGNIFICANCE: Our study provided the basis for the development of a clinical trial protocol to treat PHL.

Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells.

G protein-coupled receptors, the largest cell surface receptor family, have emerged as critical players in cell death and survival. High gene expression level of the G(q)-coupled P2Y(1) nucleotide receptor in PC-3 prostate cancer cells was demonstrated using real-time quantitative PCR and confirmed by Western blotting and confocal laser scanning microscopy. A selective P2Y(1) receptor agonist, the ADP analogue MRS2365, concentration-dependently induced intracellular calcium mobilization (EC(50) 5.28nM), which was diminished by P2Y(1) receptor-selective antagonist MRS2500. P2Y(1) receptor activation by MRS2365 induced apoptosis in assays of Caspase-3, LDH release, and annexin-V staining. The pro-apoptotic effect of MRS2365 was blocked by MRS2500, P2Y(1) siRNA, and an inhibitor of the MAP kinase pathway PD98059. MRS2365 significantly inhibited the proliferation of PC-3 cells, examined using a MTT assay. Thus, activation of the P2Y(1) receptor induced cell death and inhibited growth of human prostatic carcinoma PC-3 cells. Activation of the P2Y(1) receptor should be a novel and promising therapeutic strategy for prostate cancer.

[Establishment of a nude mouse model of highly metastatic gastric lymphoma constructed with orthotopic transplantation of surgical specimen].

OBJECTIVE: To construct a mouse model of highly metastatic gastric lymphoma with orthotopic transplantation of human primary gastric lymphoma specimen. METHODS: A fresh surgical specimen of primary gastric lymphoma was obtained intraoperatively and implanted into the submucosa of stomach in nude mice. Tumor formation, invasion, metastasis, morphological characteristics under light microscopy and electron microscopy, immunohistochemistry,and the karyotype of orthotopically transplanted tumor cells were studied. RESULTS: An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice(HGBL-0305) was successfully established. Histopathology of transplanted tumors showed primary gastric diffuse large B cell lymphoma. CD19, CD20, CD22 and CD79alpha were positive, while CD3 and CD7 were negative. The number of chromosome ranged from 56 to 69. DNA index(DI) was 1.47+/-0.12(i.e. heteroploid). Until now, HGBL-0305 model has been maintained for 45 generations by orthotopic passage for almost 4 years in nude mice. A total of 156 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumor cells were both 100%. The autonomic growth of the transplanted tumor cells invaded and destructed all the layers of the nude mice stomach. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 69.5%, 55.6%, 45.7%, and 30.5%, respectively. CONCLUSIONS: An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice is successfully established. HGBL-0305 model may simulate the natural course of primary gastric lymphoma in human and provides an ideal animal model for studies on pathogenesis, metastasis biology and anti-metastatic therapies of primary gastric lymphoma.

[Establishment of nude mice liver metastatic model of human primary malignant melanoma of the small intestine].

OBJECTIVE: To provide ideal animal models for exploring pathogenesis and experimental therapy of primary malignant melanoma of the small intestine. METHODS: The histologically intact primary and liver metastatic fragments derived from surgical specimens of one patient with metastatic malignant melanoma of the small intestine were orthotopically implanted in the small intestinal mucous layer of nude mice. The take rate, invasion and liver metastasis were observed. Morphology (light microscopy, electron microscopy), immunophenotype analysis, flow cytometry and karyotype analysis were applied for the original human tumors and the transplanted tumors. RESULTS: The primary and liver metastatic fragments of malignant melanoma of the small intestine were successfully implanted in nude mice. After continuous passages in nude mice,an orthotopic model of human primary malignant melanoma of the small intestine(from the primary focus)in nude mice (termed HSIM-0501) and a liver metastatic model of human primary malignant melanoma of the small intestine (from the liver metastatic focus) in nude mice (termed HSIM-0502) were established. Histological examination of transplanted tumors revealed high-grade melanoma. S-100 protein and HMB45 were positive. Massive melanin granules and melanin complex were seen in cytoplasm of tumor cells.Chromosomal modal number was between 55 and 59. DNA index (DI) was 1.49-1.61, representing heteroploid. HSIM-0501 and HSIM-0502 were maintained for 25 and 27 passages in nude mice respectively. Three hundred and seventeen nude mice were used for transplantation. Both the take rate after transplantation and resuscitation rate of liquid nitrogen cryopreservation were 100%. HSIM-0501 exhibited 46.2% liver metastasis and 36.7% lymph node metastases. In HSIM-0502, both liver and lymph node metastases were 100%.The transplanted tumors autonomically and invasively grew in the small intestines of nude mice and hematogenous metastasis, lymph node metastasis and celiac planting metastasis occurred. CONCLUSION: Two nude mice liver metastatic models of human primary malignant melanoma of the small intestine are successfully established, which provide ideal animal models for the research of pathogenesis,metastasis biology and anti-metastatic experimental therapy of primary malignant melanoma of the small intestine.

[AF172993 sequence of Plunc in GenBank database is not the complete CDS].

OBJECTIVE: To determine AF172993 sequence is either the complete CDS or a transcript variant. METHODS: RT-PCR was used to amplify the CDS sequence of Plunc, which was subsequently cloned into the pEGFP-N1 eukaryotic expression vector. After bi-directional sequence analysis, the sequence obtained was blasted against the AF172993 sequence, nr database and human genome database. RESULTS: In CDS of the new cloned sequence, the 658 base A in the AF172993 sequence was replaced by C, and the corresponding genetic code was also converted from AAG to CAG, leading to the alteration of the amino acid Gln to Lys. In addition, the base C at the 658 position of the CDS showed perfect match with the base C at 2094188 position in human chromosome 20. CONCLUSION: The base A at the 658 position of AF172993 sequence of Plunc is a mutation site, which alters the coding of the amino acid. AF172993 sequence is actually a transcript variant of Plunc, and the annotation to AF172993 in GenBank database is not correct and need to be revised.

[Establishment of liver metastasis model of human primary colonic lymphoma by orthotopic transplantation].

OBJECTIVE: To provide an ideal animal model for exploring pathogenesis and experimental treatment of primary colonic lymphoma. METHODS: Primary colonic and liver metastatic lymphoma tissues were obtained from the surgical specimens,and transplanted into colonic mucosa of nude mice respectively. The tumorigenesis, invasion, metastasis and morphology of the transplanted tumor were observed. Karyotype was analyzed and DNA content was measured. RESULTS: According to the new WHO classification of malignant lymphoma, two high metastatic models (HCBL-0303 from primary lymphoma and HCBL-0304 from live metastatic lesion) of human primary colonic non-Hodgkin's B cell lymphoma in nude mice were established successfully by orthotopic transplantation. Pathological examination showed poorly differentiated non-Hodgkin's B cell lymphoma of the transplanted tumors, and immunohistochemical staining showed positive expressions of CD19, CD20 and CD22, and negative expressions of CD3 and CD7. The number of chromosome ranged from 55 to 59, and DNA index (DI) was 1.59 - 1.71 (i.e. heteroploid). In HCBL-0303,liver metastasis rate was 63.7% and lymph node metastasis rate was 56.4%. However, in HCBL-0304, both metastasis rates of liver and lymph node were 100%. The transplanted tumors grew autonomously and invasively in nude mice, and further developed hematogenous, lymphatic metastasis and intraperitoneal seeding. CONCLUSIONS: HCBL-0303 and HCBL-0304 are the first established high metastatic models of primary colonic lymphoma, and can be applied to the research on pathogenesis, invasion,metastasis and experimental therapy of human primary colonic lymphoma.

Reexploring the possible roles of some genes associated with nasopharyngeal carcinoma using microarray-based detection.

In gene expression profiling, nasopharyngeal carcinoma (NPC) 5-8F cells differ from 6-10B cells in terms of their high tumorigenicity and metastatic ability. Differentially expressed genes from the two cell types were analyzed by combining with MILANO (the automatic custom annotation of microarray results which is based on all the available published work in PubMed). The results showed that five genes, including CTSD, P63, CSE1L, BPAG1 and EGR1, have been studied or mentioned in published work on NPC. Subsequently, we reevaluated the roles of these genes in the pathogenesis of NPC by combining the data of gene chips from NPCs versus NPs and pooled cells from 5-8F, 6-10B and CNE2 versus NPs. The results suggested that the roles of BPAG1 and EGR1 are possibly different from those reported in previous NPC studies. These five genes are likely to be involved in the proliferation, apoptosis, invasion and metastasis of NPC. A reexploration of the genes will further define their roles in the pathogenesis of NPC.

Liver metastasis models of human colorectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristic.

AIM:To establish a liver metastasis model of human colorectal carcinoma in nude mice.METHODS:Orthotopic transplantation of histologically intact colorectal tissues from patients into colorectal mucosa of nude mice. Tumorgenicity, invasion, metastasis and morphological characteristics of the transplanted tumors were studied by light microscopy, electron microscopy and immunohistochemistry.RESULTS:Liver metastasis models of human colon carcinoma (HCA-HMN-1) and human rectal carcinoma (HRA-HMN-2) were established after sceening from 34 colorectal carcinomas.They had been passaged in vivo for 18 and 21 generations respectively. There were lymphatic, hemotogenous and implanting metastasesis.CEA secretion was maintained after transplantation. The primary and liver metastatic tumors were similar to the original human carcinoma in histopathological and ultrastructural features, DNA content and chromosomal karyotype.CONCLUSION:The liver metastasis models provide useful tools for the study of mechanism of metastasis and its treatment of human colorectal cancer.