Production of the alpha subunit of guanine nucleotide-binding protein GO by neuroendocrine tumors

We have found that neuroendocrine tumors (including neuroblastoma, ganglioneuroma, gut carcinoid, pheochromocytoma, medullary thyroid carcinoma, insulinoma, glucagonoma, prolactinoma, carotid body tumor, and small cell lung carcinoma) produce considerable amounts (about 1000-80,000 ng/g tissue) of the alpha subunit of guanine nucleotide-binding protein, GO (GO alpha), whereas nonneuroendocrine tumors contain less than 300 ng of GO alpha/g tissue. GO alpha in the neuroendocrine tumors was present both in the soluble fraction, and cholate-extractable membrane-bound fraction of tissues. Immunoblots of membrane fractions of neuroblastoma and carcinoid tissues confirmed that the immunoreactive substance in the tumor tissues was GO alpha. Immunohistochemically, GO alpha was localized consistently in the cell membrane and occasionally in the cytoplasm of neuroendocrine tumors. GO alpha was also detected in sera of 73% patients with neuroblastoma at diagnosis, whereas serum GO alpha concentrations in control children, or patients with nonneuroendocrine tumors were lower than the detection limit of the immunoassay method employed. Serum GO alpha concentrations in patients with neuroblastoma changed with the clinical course; they fell in patients responding to treatment and increased in patients who relapsed. Since GO alpha, a specific protein in the neural and neuroendocrine cells, was found to be produced in considerable amounts by all types of neuroendocrine tumors but not in nonneuroendocrine tumors, GO alpha might be a useful biomarker for neuroendocrine tumors.     

Clonal evolution in a human neuroblastoma

Tumor samples, obtained from a single patient at two points in his illness, have enabled us to study clonal evolution in a neuroblastoma. Cells from the primary tumor demonstrated considerable heterogeneity in terms of chromosome number; cells from 4 subsequent metastases were all nearly diploid; and cells from a tumor produced in a mouse by the injection of cells from the primary tumor were hypotriploid in modal number. All of the tumor samples contained the same marker chromosome rearrangements, indicating their origin from a common precursor. Each of the cell lines analyzed (including those from the patient's metastases, those from the tumor in a mouse, and those from the primary tumor after 11 months in continuous culture) also contained different and distinguishing chromosome abnormalities. The differences in karyotype among these tumor samples and cell lines presumably reflect the different selection pressures at work in each instance.     

Expression and distribution of peripherin protein in human neuroblastoma cell lines

A series of human neuroblastoma (NB) cell lines was analyzed for expression of peripherin, a class-III intermediate filament protein expressed at high levels in ganglia of the peripheral nervous system. By Western blotting, peripherin protein was detected in all human NB cell lines examined. The highest level of peripherin was found in the NUB-7 cell line, previously characterized as homogeneously neuroblastic. By immunofluorescence labeling, peripherin was shown to be organized in a perinuclear filamentous pattern and, exemplified by IMR32 cells, was also shown to be localized to spontaneously formed neurites. Peripherin was expressed in neuroblastic but not substrate-adherent cells, and was found at low levels in I-type cells. There was a pronounced redistribution of peripherin to neurites formed in response to dibutyryl cyclic adenosine monophosphate (dbcAMP) and all-trans-retinoic acid (RA). In NUB-7 cells, which do not extend neurites in response to nerve growth factor, there was no change in the level of peripherin protein following treatment with this agent. Both dbcAMP and RA induced a redistribution of peripherin to neurite extensions, but only treatment with RA increased the level of the protein as demonstrated with NUB-6A4 and NUB-6C4 subclones. Peripherin was also variably expressed in peripheral neuroepithelioma (NE) cell lines tested, but was organized into a more basket-like filamentous pattern in these cells. The heterogeneous expression and distribution of peripherin in NB and NE cell lines indicate that this protein is associated with maturation of the neuronal phenotype and hence serves as a differentiation marker for tumors derived from the neural crest.     

Constitutive activation of stimulatory guanine nucleotide binding protein (G(S)alphaQL)-mediated signaling increases invasiveness and tumorigenicity of PC-3M prostate cancer cells.

An abnormal stimulation of cAMP signaling cascade has been implicated in various human carcinomas. Since the agents activating G(S)alpha-mediated signaling pathways have been shown to increase in vitro proliferation of prostate cancer cells, present studies examined the G(S)alpha-mediated signaling in tumorigenicity and invasiveness of PC-3M prostate cancer cells. PC-3M cells were stably transfected with plasmids containing either wild type (G(S)alpha-WT) or constitutively active (gsp mutant of G(S)alpha or G(S)alpha-QL) cDNAs. The stable transfectants were then tested for: (1) colony formation in soft agar; (2) cell migration and penetration of basement matrix in an in vitro invasion assay; and (3) the ability to form tumors and metastases in nude mice. PC-3M cells expressing G(S)alpha-QL protein displayed 15-fold increase in their ability to migrate and penetrate the basement membrane as compared to parental PC-3M cells or those expressing G(S)alpha-WT. G(S)alpha-QL transfectants also displayed a dramatically greater rate of growth in soft agar, and greater tumorigenicity and metastasis forming ability when orthotopically implanted in nude mice. All mice receiving PC-3M cells produced primary tumors within 5 weeks after implantation. However, the cells expressing G(S)alpha-QL displayed a significantly faster tumor growth as assessed by prostate weight (greater than 20-fold as compared to PC-3M cells), and produced metastases in kidneys, lymph nodes, blood vessels, bowel mesentery and intestine. Interestingly, expression of G(S)alpha-WT reduced the ability of PC-3M cells to form tumors in nude mice. These results suggest that persistent activation of G(S)alpha-mediated signaling cascade can dramatically accelerate tumorigenesis and metastasizing ability of prostate cancer cells.     

勃起功能障碍的药物治疗

为理解现代药物治疗勃起障碍的作用机制,首先要注意2个重要的器官作为实现勃起状态的前提:阴茎海绵体作为自主调控器官需接受由大脑传出的冲动(有时也通过脊髓反射),阴茎由萎软变膨大坚挺;另一方面,阴茎本身可看作是一活跃的、由平滑肌细胞组成的海绵状物,由自律的神经支配诱发平滑海绵状肌细胞等长舒张,海绵体膨胀变大并通过外周阻力的降低出现显著血流增加及持续充血状态.     

Identification of a novel activity of human papillomavirus type 16 E6 protein in deregulating the G1/S transition

In this study we show that E6 of human papillomavirus has the ability to deregulate the cell cycle G1/S transition. In rodent immortalized fibroblasts (NIH3T3) serum deprivation or over-expression of the cyclin-dependent kinase inhibitors, p16(INK4a) or p27(KIP1), leads to G1 cell cycle arrest. HPV16 E6 overcomes the antiproliferative signals, gaining the ability to drive serum-deprived and p16(INK4a) or p27(KIP1) over-expressing cells into S phase. E6 protein from the benign HPV type 1 displays a similar activity to HPV16 E6 to deregulate the G1/S transition. Thus, this activity appears to be conserved between E6 proteins from non-oncogenic and oncogenic HPV types. Furthermore, we show that HPV16 E6 is not able to circumvent a G1 arrest imposed by pRb mutant in which all CDK phosphorylation sites have been mutated. These data indicate that the viral protein acts upstream of pRb and its mechanism in promoting cell cycle progression is dependent on pRb phosphorylation. In summary, this study describes a novel biological function of HPV E6 and shows that the S phase entry, required for viral DNA replication, is not exclusively controlled by E7, but that E6 also is involved in this event.     

Expression of the smg p25A (a ras p21-like GTP-binding protein) gene in human neuroblastoma cell lines and tumor tissues

We have examined expression of the smg p25A (a ras p21-like GTP-binding protein) gene in neural crest-derived tumor cell lines and neuroblastoma tissues. The human neuroblastoma cell lines GOTO, IMR-32, NB-1, and SK-N-SH expressed the 1.6-kilobase smg-25A mRNA. SH-SY5Y and SH-IN, variant cell lines with a neuronal phenotype derived from SK-N-SH, expressed much more smg-25A mRNA than did SH-EP1, a variant line with an epithelium-like phenotype also derived from SK-N-SH. The primitive neuroectodermal tumor cell lines SK-N-MC and KU-SN and the Ewing's sarcoma cell lines RD-ES and SK-ES expressed the smg-25A mRNA to a much smaller extent than did neuroblastoma cell lines. Of 15 human neuroblastoma specimens tested, 13 expressed the smg-25A mRNA to various extents. When the relative ratio of the smg-25A mRNA level to the glyceraldehyde-3-phosphate dehydrogenase mRNA level was compared among neuroblastoma tumor tissues, the value was significantly higher in tumors histologically diagnosed as ganglioneuroblastoma. The smg-25A mRNA was not detected in the tissues of Hodgkin's lymphoma, Wilms' tumor, Ewing's sarcoma, or undifferentiated sarcoma of the liver. These results suggest that the smg-25A mRNA level is closely related to the neuronal differentiation state of tumors derived from the neural crest.     

Biological properties of a tumour cell line (NB1-G) derived from human neuroblastoma

The properties of a new tumour cell line (NB1-G) derived from human neuroblastoma by xenografting in nude rats followed by adaptation to tissue culture are described. Studies using a panel of monoclonal antibodies demonstrate the neuro-ectodermal nature of the cells and support the diagnosis of the primary tumour as neuroblastoma. Cytogenetic studies have revealed a human karyotype with several chromosomal abnormalities. Genetic analysis by in situ DNA hybridization has demonstrated the presence of multiple copies of the N-myc gene. Approximately 20-30 fold amplification of the gene is observed on Southern blot analysis. The cell line has been adapted to growth as multicellular tumour spheroids as well as monolayer culture. Radiobiological studies on spheroids show the cells to be radiosensitive with low capacity for sub-lethal damage accumulation and repair. The cell line should be useful for fundamental studies of human neuroblastoma as well as experimental therapy in vitro.     

Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model

The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (ATase) is a major component of resistance to treatment with methylating agents and nitrosoureas. Inactivation of the protein, via the administration of pseudosubstrates, prior to chemotherapy has been shown to improve the latter's therapeutic index in animal models of human tumours. We have also shown that rational scheduling of temozolomide, so that drug is administered at the ATase nadir after the preceding dose, increases tumour growth delay in these models. We now report the results of combining these two approaches. Nude mice bearing A375M human melanoma xenografts were treated with vehicle or 100 mg/kg temozolomide ip for 5 doses spaced 4, 12 or 24 hr apart. Each dose was preceded by the injection of vehicle or 20 mg/kg 4BTG. All treatments resulted in significant delays in tumour quintupling time compared with controls: by 6.2, 5.9 and 16.8 days, respectively, for 24-, 12- and 4-hourly temozolomide alone and by 22.3, 21.3 and 22.1 days, respectively, in combination with 4BTG. Weight loss due to TMZ was unaffected by the presence of 4BTG. This was of the order of 6.2-10.6% with 24- and 12-hourly administration and 17.4-20.1% (p < 0.0001) with 4-hourly treatment. In our model, combining daily temozolomide with 4-BTG confers increased antitumour activity equivalent to that achieved by compressing the temozolomide schedule but with less toxicity. Using temozolomide schedule compression with 4-BTG does not improve on this result, suggesting that ATase inactivation with pseudosubstrates is a more promising means of enhancing the activity of temozolomide than compressed scheduling.     

Meta-iodobenzylguanidine (mIBG) uptake and storage in the human neuroblastoma cell line SK-N-BE(2C)

Whilst many human neuroblastoma cell lines have been studied to see if they are capable of taking up mIBG, few appear to have this ability. This contrasts markedly to the situation in vivo, where uptake has been demonstrated in the majority of tumours investigated. Here we report on the human neuroblastoma cell line SK-N-BE(2C) and demonstrate that mIBG uptake can occur in this cell line through 2 mechanisms. At low concentrations of mIBG (approximately 10(-8) M) an active transport process predominates, whereas at non-physiological levels (10(-4) M) uptake occurs through passive diffusion. The active transport process is ATP-, Na(+)- and temperature-dependent. Uptake is blocked by 10(-6) M desipramine, an inhibitor of the uptake-I mechanism involved in amine transport. In contrast, desipramine has no effect on the passive diffusion of mIBG into cells. The active transport mechanism for mIBG uptake appears rather promiscuous for biogenic amines, as dopamine, tyramine and nor-adrenaline were highly efficient at blocking mIBG entry to the cell. Serotonin and histamine were capable of interfering with mIBG uptake only at much higher concentrations. Electron microscopy of SK-N-BE(2C) cells revealed a paucity of neurosecretory granules. Biochemical investigations demonstrated the majority of mIBG to be present in the cytoplasm of cells. The availability of a human neuroblastoma cell line that grows well, both as xenograft and in culture, should further our understanding of the cytotoxic effects of mIBG and thus enhance its clinical usefulness.     

Verapamil enhancement of antitumor effect of cis-diamminedichloroplatinum(II) in nude mouse-grown human neuroblastoma

The antitumor effect of combined use of cis-diamminedichloroplatinum(II) (CDDP) and verapamil, a calcium influx blocker, was examined in neuroblastoma transplanted to BALB/c athymic mice. The response of the tumor to CDDP was related to the dose administered. Regression of the tumor was observed when CDDP was administered at 4.2 mg/kg/injection. The retardation of tumor growth was observed in the group to which CDDP was administered at 2.1 mg/kg/injection. When verapamil was administered with CDDP, regression of the tumor was observed in the group treated with CDDP at 2.1 mg/kg/injection, and the retardation of tumor growth was observed in the group treated with CDDP at 1.4 mg/kg/injection. These results indicate that verapamil enhances the antitumor effect of CDDP against transplanted neuroblastoma in BALB/c athymic mice.     

Identification of a multidrug resistance associated antigen (P-glycoprotein) in normal human tissues

The multidrug resistance (MDR) phenotype describes a pattern of cross-resistance to unrelated compounds observed in mammalian cell lines selected in vitro for resistance to a single agent. Overexpression of a 170 000 dalton cell membrane glycoprotein (P-glycoprotein) is associated consistently with this phenotype in these cell lines. Recently, several human tumours have been shown to contain P-glycoprotein and expression was greatest in tumours exhibiting clinical drug resistance. To explore further the significance of P-glycoprotein, we examined normal human tissues obtained at autopsy by polyacrylamide gel electrophoresis and immunoblotting using a monoclonal antibody directed against P-glycoprotein. We showed expression of P-glycoprotein in normal liver and small bowel mucosa but not in other organs examined. This suggests there may be significant expression of P-glycoprotein in certain normal human tissues and any plan to exploit P-glycoprotein clinically must take these findings into account.     

Role of a proteolysis-inducing factor (PIF) in cachexia induced by a human melanoma (G361).

Human melanoma, G361, which induces cachexia in nude mice, has been shown to produce a proteolysis-inducing factor (PIF) of Mr 24000, which is immunologically identical to that isolated from a cachexia-inducing murine tumour (MAC16). Biosynthetic labelling of G361 cells using a combination of [35S]sulphate and [6-3H]glucosamine gave a single component of Mr 24000 after affinity chromatography employing a murine monoclonal antibody. The material contained both radiolabels and, after digestion with peptide N-glycosidase F, two fragments were produced of Mr 14000 and 10000 also containing both radiolabels. Digestion with O-glycosidase produced three fragments of Mr 14000, 6000 and 4000, the first two of which contained both radiolabels, while the third only contained 3H. This digestion pattern is the same as that previously observed with PIF from the MAC16 tumour and is commensurate with one N-linked sulphated oligosaccharide chain of Mr 10000, one O-linked sulphated oligosaccharide chain of Mr 6000 and a central polypeptide chain of Mr 4000 with some residual carbohydrate. When PIF from G361 cells was administered to female NMRI mice (20 g) a pronounced depression of body weight (1.36+/-0.36 g; P < 0.0001 from control) was observed over a 24 h period without a decrease in either food or water consumption. Body composition analysis showed a significant decrease in the non-fat carcass mass without a change in carcass fat or body water. This result suggests that depletion of lean body mass in mice bearing G361 melanoma arises from the production of PIF.     

Morphological differentiation of human neuroblastoma cell lines by a new synthetic polyprenoic acid (E5166)

The prognosis of patients with advanced neuroblastoma remains poor despite recent progress in chemo/radiotherapy. Therapeutic trials on the induction of differentiation of neuroblastoma by chemical and biological agents have been attempted to improve patients' prognosis. Recently a new synthetic polyprenoic acid, E5166, having retinoic acid properties, has been described. In this study two human neuroblastoma cell lines, KP-N-RT(LN) and SK-N-DZ, were treated in vitro by E5166. Morphological differentiation of KP-N-RT(LN) and SK-N-DZ cells could be induced by E5166 in the presence of 1.7 X 10(-5) M E5166 for 10 days in culture. Levels of catecholamines (dopamine, adrenaline, and noradrenaline) were not elevated in the E5166-differentiated cells. E5166-induced differentiation may not be cyclic AMP dependent, since levels of cyclic AMP did not increase after exposure of cells to this agent. No significant increase in neuron-specific enolase levels could be demonstrated in E5166-treated neuroblastoma as compared to control untreated cells. E5166 treatment of KP-N-RT(LN) and SK-N-DZ cells was found to inhibit colony formation in soft agar in a dose-dependent manner. Colonies of KP-N-RT(LN) cells in the presence of E5166 showed morphological differentiation as defined by the expression of long neurite processes. E5166 is a less toxic reagent than the retinoic acids used for the induction of differentiation, it can be administered to patients p.o., and the concentration of E5166 which induces the morphological differentiation in vitro can be achievable in vivo. Therefore our study suggests that E5166 could be a useful therapeutic agent in advanced neuroblastoma to differentiate residual anaplastic tumor cells to a benign form (ganglioneuroma) after surgery and chemotherapy.     

G protein pathway suppressor 2 (GPS2) acts as a tumor suppressor in liposarcoma

Liposarcoma(LPS) is the most common type of soft tissue sarcoma accounting for 20 % of all adult sarcomas. However, the molecular pathogenesis of this malignancy is still poorly understood. Here, we showed that GPS2 expression was downregulated in LPS and correlated with the prognosis of this disease. In vitro study showed that knockdown of GPS2 resulted in enhanced proliferation and migration of LPS cell line SW872, without significant influence of cell death. Conclusively, our results suggest that GPS2 may act as a tumor suppressor in LPS and serve as a potential prognosis marker for this disease.

     

Identification of the cellular protein encoded by the human Wilms' tumor (WT1) gene.

A putative tumor-suppressor gene (wt1) located at chromosome 11p13 and involved in Wilms' tumor development has recently been identified as a zinc finger polypeptide-encoding gene. The purpose of this study was to characterize the protein encoded by the human wt1 gene. The region spanning the entire zinc finger domain was amplified by polymerase chain reaction (PCR) and subcloned in the pATH 3 expression vector. Polyclonal antibodies against the fused TrpE-WT protein were raised. These antibodies immunoprecipitated a 49- to 51-kDa protein from hematopoietic tumor cells labeled in vivo with [35S]methionine. Subcellular fractionation and immunohistochemistry followed by confocal microscopy indicated that the Wilms' tumor gene product (WT1) is mainly localized within the nucleus.     

Identification of tumorigenic cells in Kras(G12D)-induced lung adenocarcinoma

We established an inducible Kras(G12D)-driven lung adenocarcinoma in CCSP-rtTA/TetO-Cre/LSL-Kras(G12D) mice that enable pursuits of the cellular and molecular processes involved in Kras-induced tumorigenesis. To investigate the cellular origin of this cancer, we first report a strategy using fluorescence-activated cell sorting fractionation that could highly enrich bronchiolar Clara and alveolar type II cells, respectively. The EpCAM(+)MHCII(-) cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than EpCAM(+)MHCII(+) cells (alveolar origin) in primary tumors that had been already initiated with oncogenic Kras activation. In addition, secondary tumors derived from EpCAM(+)MHCII(-) cells showed diversity of tumor locations compared with those derived from EpCAM(+)MHCII(+) cells. In the alveolar region, secondary tumors from EpCAM(+)MHCII(-) cells expressed not only bronchiolar epithelial marker, panCK, but also differentiation marker, proSPC, consistent with the notion that cancer-initiating cells display not only the abilities for self-renewal but also the features of differentiation to generate heterogeneous tumors with phenotypic diversity. Furthermore, high level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM(+)MHCII(-) cells. Therefore, these results suggest that bronchiolar Clara cells are the origin of cells and tumorigenesis for Kras(G12D)-induced neoplasia in the lungs.