Neoplastic Cell Lines Research Articles

Design, synthesis, and biological evaluation of novel pyridine acid esters of podophyllotoxin and esters of 4′-demethylepipodophyllotoxin

Podophyllotoxin and related analogs present numerous challenges associated with optimal antitumor activity and severe unpredictable toxicity. In the course of our ongoing investigation of quantitative structure–activity relationships to find biorational antitumor drugs, two series of pyridine acid ester derivatives of podophyllotoxin and 4′-demethylepipodophyllotoxin have been prepared by reacting the corresponding pyridine acids with the hydroxyl group of podophyllotoxin and 4′-demethyl epipodophyllotoxin in the presence of dimethylaminopyridine (DMAP) and N,N-dicyclohexylcarbodiimde (DCC). The structures of the compounds were extensively characterized by using 1H-nuclear magnetic resonance, mass spectroscopy, infrared, and elemental analysis and evaluated for their in vitro cytotoxicity on two neoplastic cell lines (P-388 murine leukemia, A-549 human lung carcinoma) using a MTT-based assay, the results indicated significantly higher efficacy of these compounds in comparison with the prototypical inhibitor etoposide. On the basis of the preliminary biological testing results, it suggested that the cytotoxic activity of OCH3 at C-4′ is more potent than that of its demethylated analogs and the podophyllotoxin substitution at C-4 may be optimal for synthesizing more potent cytotoxic compounds.

Paracrine Suppression of VEGF Secretion by Erythropoietin Inhibits Tumor Growth.

Several studies have reported that erythropoietin (Epo) is a pleiotropic cytokine with biological properties in addition to its primary function in regulating maturation, growth and survival along the erythroid lineage. Recently, a number of investigators have reported that various neoplastic tissues and human cancer cell lines express Epo and the Epo receptor (EpoR), raising suspicion for the presence of an autocrine-paracrine Epo-EpoR system. It has been shown that inhibition of vascular endothelial growth factor (VEGF) results in an increase of Epo secretion and increased hematocrit in vivo. In this study, we used an in vivo Lewis lung carcinoma model to examine a converse Epo effect on VEGF production and metastasis. Lewis lung carcinoma (LLC) cells were injected subcutaneously into C57BL mice. The plasma levels of VEGF, the tumor vessel formation, the size of the primary tumors and the extent of lung metastatic disease were determined. In addition, intravenously injected LLC cells seeded in the lungs were assessed. Tumor-bearing animals treated with Epo had 23.6% less VEGF in the plasma compared to saline treated mice (p<0.04). There was no correlation between VEGF concentration and hemoglobin levels in either group of animals. Tumor sections indicated that the number of blood vessels was higher (10.7% for inner and 23.8% for outer, respectively) in tumors obtained from animals treated with saline compared to Epo-treated mice (p>0.05). Using non-parametric analysis, we found that there was a statistically significant difference in tumor growth between saline-treated and Epo-treated animals (p<0.05). However, the number of lung metastases derived from primary tumors was similar in both groups. In assessing size of the metastatic tumors, we found that the average volume of lung nodules was 24.2% higher in saline-injected animals compared to Epo-treated mice. The number of tumors seeded in the lungs following intravenous injection of LLC cells was similar in animals treated with a high dosage of Epo, low dosage of Epo or saline. In addition, the average volume of the nodules was reduced by 42% in animals treated with high and low concentrations of Epo compared to the control group (p = 0.03). In conclusion, Epo exerts a paracrine suppressive effect on VEGF secretion resulting in slower tumor growth in this model.

High expression of CEACAM6 and CEACAM8 mRNA in acute lymphoblastic leukemias

CEACAM family members are a set of widely expressed proteins involved in several biological functions, including cell adhesion, migration, signal transduction, and the regulation of gene expression. Abnormal overexpression and downregulation of some CEACAMs have been described in tumor cells. Monoclonal antibodies grouped in the CD66 cluster recognize CEACAM members. Ectopic CD66 expression is commonly detected in B-cell lineage acute lymphoblastic leukemia (ALL). To investigate the CEACAM messenger RNA (RNA) expression in leukemic blasts, we performed a quantitative polymerase chain reaction (RQ-PCR) analysis in purified RNA samples from a consecutive series of acute leukemias (135 patients). Most B-cell lineage ALL expressed CD66 (79.5%), whereas no single case of T-cell lineage ALL disclosed CD66 reactivity (0%). All the BCR-ABL+ ALL cases showed CD66 expression. CD66 was positive even in cases without CD10 expression (72.7%) and/or with MLL rearrangements. Despite the sharp contrast between T-ALL and B-ALL in CD66 reactivity, CEACAM patterns were comparable, and only minor differences for CEACAM1 and CEACAM8 were detected. All the leukemic samples showed overexpression of CEACAM6 and 8 when compared with normal granulocytes. These results were confirmed by dilutional experiments. The leukemic pattern paralleled the normal regenerating bone marrow with lower values for CEACAM1. In line with the results for CD66 reactivity, neoplastic cell lines had a uniform low expression of CEACAM family members. It remains to be investigated whether these CEACAM disturbances provide growth advantages to tumoral cells by inhibiting the anoikis process.

Carnitine palmitoyltransferase I in human carcinomas: A novel role in histone deacetylation?

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for β-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells.We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n=11) and breast carcinomas (n=24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p≤0.04), whereas FAS was increased (p≤0.04). A striking CPT1 nuclear localization was evident in the tumors (p≤0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins co-immunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1.The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

MCL-1 as a Buffer for Proapoptotic BCL-2 Family Members during TRAIL-induced Apoptosis: A MECHANISTIC BASIS FOR SORAFENIB (BAY 43-9006)-INDUCED TRAIL SENSITIZATION

Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-x(L) as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-x(L) but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(N(alpha)-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.

Divergent Synthesis of Cytotoxic Styryl Lactones fromd-Xylose. The First Total Synthesis of (+)-Crassalactone C

A new divergent approach to (+)-goniofufurone (1) and 7-epi-(+)-goniofufurone (2), as well as the first total synthesis of crassalactone C (3), has been achieved starting from D-xylose. In a preliminary bioassay, all three natural products 1, 2, and 3 showed remarkable in vitro antiproliferative activities against K562, Raji, and HeLa neoplastic cell lines.

Design, synthesis, and biological evaluation of novel spin-labeled derivatives of podophyllotoxin as potential antineoplastic agents. Part XII¶

Five novel nitroxyl spin-labeled ester derivatives of podophyllotoxin (11a–11e) have been prepared and their structural information on these nitroxyl spin-labeled ester derivatives of podophyllotoxin (11a–11e) using 1HNMR spectroscopy was efficiently obtained by application of the in situ reduction of representative nitroxyl spin-labeled ester derivative of podophyllotoxin 11e with phenylhydrazine for the preparation of N-hydroxylamine 12 in the NMR tube. These novel derivatives were further evaluated for their in vitro cytotoxic activity against five neoplastic cell lines (K562, HL-60, SPCA-1, Lewis, and L-1210) using MTT assay. Most of the target compounds (except for all these compounds against SPCA-1) exhibited more pronounced cytotoxicity against several neoplastic cell lines than that of the prototypical inhibitor etoposide. ¶For part XI, see Jin, Y., Chen, S. W., and Tian, X. Bioorg. Med. Chem. 2006, 14, 3062-3068.

Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells

Although a number of studies have shown that vitamin K possesses antitumor activities on various neoplastic cell lines, there are few reports demonstrating in vivo antitumor effects of vitamin K, and the antitumor effect on colorectal cancer (CRC) remains to be examined. Therefore, antitumor effects of vitamin K on CRC were examined both in vitro and in vivo. Vitamins K2, K3 and K5 suppressed the proliferation of colon 26 cells in a dose-dependent manner, while vitamin K1 did not. On flow cytometry, induction of apoptosis by vitamins K2, K3 and K5 was suggested by population in sub-G1 phase of the cell cycle. Hoechst 33342 staining and a two-color flow cytometric assay using fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that vitamins K2, K3 and K5 induced apoptotic death of colon 26 cells. Enzymatic activity of caspase-3 in colon 26 cells was significantly up-regulated by vitamins K2, K3 and K5. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, substantially prevented vitamin K-mediated apoptosis. In vivo study using syngeneic mice with subcutaneously established colon 26 tumors demonstrated that intravenous administration of vitamins K2, K3 and K5 significantly suppressed the tumor growth. The number of apoptotic tumor cells was significantly larger in the vitamin K-treated groups than in the control group. These results suggest that vitamins K2, K3 and K5 exerted effective antitumor effects on CRC in vitro and in vivo by inducing caspase-dependent apoptotic death of tumor cells, suggesting that these K vitamins may be promising agents for the treatment of patients with CRC.

The Hsp32/HO-1-targeted drug SMA-ZnPP counteracts the proliferation and viability of neoplastic cells in solid tumors and hematologic neoplasms

14122 Background: Heat shock protein 32 (Hsp32) is a stress-related survival factor that is overexpressed in various neoplastic cells. Recently, specific Hsp32- targeting drugs such as styrene maleic acid encapsulated zinc protoporphyrin (SMA-ZnPP) have been developed. Methods: We examined the effects of SMA-ZnPP on proliferation and survival of various tumor cell-lines, including U97MG (glioblastoma), A549 (lung cancer), MDA-MB-231 (breast cancer), BxPC-3 (pancreatic), HepG2 (hepatocellular), Colo201, Colo320DM, DLD-1 (colon), OvCar3 (ovarian carcinoma), KG1, U937, HL60, K562 (myeloid leukemias), RAJI, NALM-6 (lymphatic leukemias), RPMI 8226, U266 (multiple myeloma) as well as on primary neoplastic cells. Moreover, Ba/F3 cells with doxycycline-inducible expression of oncoproteins (RAS-G12V, BCR/ABL, KIT-D816V) were analyzed. Expression of Hsp32 mRNA was examined by RT-PCR and Northern blotting, and expression of the Hsp32 protein by Western blotting. To silence Hsp32 in neoplastic cells, we used specific siRNA as well as SMA-ZnPP. Proliferation was analyzed by 3H-thymidine uptake and apoptosis by light microscopy. Results: All neoplastic cells tested were found to express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. In Ba/F3 cells, induction of RAS-G12V, BCR/ABL, or KIT D816V enhanced the expression of Hsp32. The Hsp32 siRNA was found to lead to a reduced viability and induction of apoptosis. Treatment of malignant cells with SMA-ZnPP resulted in a significant decrease in proliferation and induction of apoptosis. The effects of SMA- ZnPP on primary neoplastic cells and cell lines were dose-dependent and occurred at pharmacologic concentrations (IC50 1–30 μM). Moreover, SMA-ZnPP was found to synergize with various anti-neoplastic drugs (cisplatin, cytarabine, tyrosine kinase inhibitors, bortezomib) in producing growth-inhibition in neoplastic cells. Conclusions: The Hsp32-targeting drug SMA-ZnPP counteracts malignant cell growth and sensitizes neoplastic cells against various other targeted or conventional antineoplastic drugs. Hsp32-targeting drugs may represent an interesting new aproach to inhibit malignant cell growth in solid tumors and leukemias. No significant financial relationships to disclose.

Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor–β1‐mediated regulatory abnormalities including up‐regulation of C‐Myc and MTA1

Although it is known that small intestinal carcinoids are derived from enterochromaffin (EC) cells, these cells remain poorly characterized and little is known of the growth regulatory mechanisms of these neuroendocrine cells. Down-regulation or loss of the transforming growth factor-beta1 (TGFbeta1) cytostatic program and activation of TGFbeta-mediated transcriptional networks is associated with uncontrolled growth and metastasis in other neural tumors, glioblastomas. Whether this phenomenon is common to small intestinal carcinoid tumors was investigated. The effects of TGFbeta1 on cultured normal EC cells (isolated by FACS sorting) and the neoplastic EC cell line, KRJ-I, was assessed using the MTT assay. The TGFbetaRII transcript and protein were identified in tumor cells and the effects of TGFbeta1 on SMAD2 phosphorylation and nuclear translocation quantified. The time-dependent response of SMAD4, SMAD7, c-Myc, and P21(WAF1/CIP1) protein expression and c-Myc and p21(WAF1/CIP1) transcript was measured in response to TGFbeta1 and the transcript expression of candidate downstream targets, MTA1 and E-cadherin, were assessed. TGFbeta1 inhibited normal EC cell proliferation (IC(50) = 17 pM) but stimulated neoplastic EC cell proliferation (EC(50) = 22 pM). In tumor cells, significantly decreased transcript (P < .01) of TGFbetaRII was identified, but no receptor mutations were identified and protein expression was evident. TGFbeta1 (1 ng/mL) resulted in SMAD2 phosphorylation and <7% nuclear expression compared with 93% in normal EC cells. In neoplastic cells, TGFbeta1 (1 ng/mL) caused a decrease in SMAD4 (>16%, P < .05), whereas SMAD7 and c-Myc transcript and protein were respectively increased >21% (P < .05) and approximately 40% (P < .002). TGFbeta1 (1 ng/mL) also decreased p21(WAF1/CIP1) transcript by 60% (P < .001) and protein that was undetectable at 24 hours. Expression of the downstream targets of the c-Myc pathway, MTA1, was increased (20%) and E-cadherin decreased (30%). The neoplastic EC cell is characterized by loss of TGFbeta-1-mediated growth inhibition and, similar to glioblastomas, utilizes the TGFbeta system to induce gene responses associated with growth promotion (c-Myc and the ERK pathway), invasion (E-cadherin), and metastasis (MTA1).

Neoplastic and Non-neoplastic Cell Lines from a Malignant Peripheral Nerve Sheath Tumour of the Cervix of a Rat

A homotransplantable tumour (LY) and cell lines (LY-PPB6 and LY-H12) were established from a spontaneous malignant peripheral nerve sheath tumour (PNST) of the uterine cervix of an F344 rat. Primary and LY tumours consisted of oval or spindle-shaped cells arranged in a flatfield or streaming fashion, and indistinct nuclear palisades were seen. Immunohistochemically, neoplastic cells reacted to vimentin, S-100 protein, neuron-specific enolase (NSE), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) in varying degrees, indicating neurogenic derivation. LY-PPB6-induced tumours in syngeneic rats developed cellular whorling patterns reacting particularly strongly to S-100 protein, NSE, MBP and GFAP. Nerve growth factor (NGF) mRNA expression was shown in LY-PPB6 cells by the reverse transcription-polymerase chain reaction (RT-PCR). By contrast, LY-H12 had a normal chromosomal number of 42, and did not produce tumours when injected into syngeneic rats. LY-H12 cells reacted to vimentin and alpha-smooth muscle actin (alpha-SMA), and the alpha-SMA-positive cell number was increased dose-dependently by the addition of transforming growth factor (TGF)-beta1, indicating a myofibroblastic nature. LY-PPB6 cells were neoplastic with properties of PNS cells, whereas LY-H12 cells were non-neoplastic stromal cells showing myofibroblastic differentiation. As TGF-beta1 mRNA expression was shown in both LY-PPB6 and LY-H12 cells by the RT-PCR, the myofibroblastic phenotype of LY-H12 cells may be mediated by paracrine or autocrine signalling in tumour tissues. LY-PPB6 and LY-H12 may prove useful for studies on the pathobiological nature of neoplastic cells and interactions between neoplastic and stromal cells in PNSTs.

Paradoxical roles for lysyl oxidases in cancer—A prospect

Lysyl oxidase (LOX) is an extracellular matrix (ECM) enzyme that catalyzes the cross-linking of collagens or elastin in the extracellular compartment, thereby regulating the tensile strength of tissues. However, recent reports have demonstrated novel roles for LOX, including the ability to regulate gene transcription, motility/migration, and cell adhesion. These diverse functions have led researchers to hypothesize that LOX may have multiple roles affecting both extra- and intracellular cell function(s). Particularly noteworthy is aberrant LOX expression and activity that have been observed in various cancerous tissues and neoplastic cell lines. Both down and upregulation of LOX in tumor tissues and cancer cell lines have been described, suggesting a dual role for LOX as a tumor suppressor, as well as a metastasis promoter gene--creating a conundrum within the LOX research field. Here, we review the body of evidence on LOX gene expression, regulation, and function(s) in various cancer cell types and tissues, as well as stromal-tumor cell interactions. Lastly, we will examine putative mechanisms in which LOX facilitates breast cancer invasion and metastasis. Taken together, the literature demonstrates the increasingly important role(s) that LOX may play in regulating tumor progression and the necessity to elucidate its myriad mechanisms of action in order to identify potentially novel therapeutics.

Smallest Peptoids with Antiproliferative Activity on Human Neoplastic Cells

Libraries of new, small peptoid monomers and dipeptoids were synthesized and assayed for antiproliferative activity against representative human neoplastic cell lines. The C-terminal N-alkyl amide peptoids are cytotoxic and are the smallest peptoids reported to have such activity. These compounds were conveniently synthesized on a BAL resin. Owing to their structure, the peptoids did not suffer from DKP formation, a problematic side reaction typically observed in peptide and peptoid synthesis.

The oncoprotein LMO2 is expressed in normal germinal-center B cells and in human B-cell lymphomas

AbstractWe previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

Transcriptional Profiling of Hematologic Malignancies with a Low-Density DNA Microarray

High-density microarrays are powerful tools for expression analysis of thousands of genes simultaneously; however, experience with low-density microarrays in gene expression studies has been limited. We developed an optimized procedure for gene expression analysis based on a microarray containing 538 oligonucleotides and used this procedure to analyze neoplastic cell lines and whole-blood samples from healthy individuals and patients with different hematologic neoplasias. Hierarchical clustering and the Welch t-test with adjusted P values were used for data analysis. This procedure detects 0.2 fmol of mRNA and generates a linear response of 2 orders of magnitude, with CV values of <20% for hybridization and label replicates. We found statistically significant differences between Jurkat and U937 cell lines, between blood samples from 15 healthy donors and 59 chronic lymphocytic leukemia (CLL) samples, and between 6 acute myeloid leukemia patients and 4 myelodysplastic syndrome patients. A classification system constructed from the expression data predicted healthy or CLL status from a whole-blood sample with a 97% success rate. Transcriptional profiling of whole-blood samples was carried out without any cellular or sample manipulation before RNA extraction. This gene expression analysis procedure uncovered statistically significant differences associated with different hematologic neoplasias and made possible the construction of a classification system that predicts the healthy or CLL status from a whole-blood sample.

The transcription factor ATF5 is widely expressed in carcinomas, and interference with its function selectively kills neoplastic, but not nontransformed, breast cell lines

ATF5, a transcription factor important in differentiation, proliferation and survival, has been found to be highly expressed in neural progenitor cells and in certain tumors including glioblastomas (GBMs), but its expression in other normal and neoplastic tissues has not been extensively investigated. A tissue microarray immunostained for ATF5 showed diffuse nuclear expression (as defined by the presence in greater than 25% of cells) in 63% (117/186) of neoplastic samples, when compared to only 32% (20/62) in nonneoplastic tissues. When analyzed by histologic subtype, a significantly greater proportion of adenocarcinomas, transitional cell carcinomas, squamous cell carcinomas and metastatic carcinomas of various tissue origins had nuclear staining when compared to nonneoplastic tissues. There was no significant difference in ATF5 expression in renal cell carcinomas, lymphomas and seminomas, when compared to nonneoplastic tissues. An expanded series of nonarray breast resection specimens revealed a significantly greater proportion of ATF5 positivity in ductal and lobular carcinomas, when compared to normal breast tissue. Past work found that loss of ATF5 function triggers death of GBM cells, but not of normal activated astrocytes. Here, we observed that loss of ATF5 function caused significant apoptotic death of neoplastic breast cell lines, but not of nonneoplastic breast cell lines. Our data demonstrate elevated ATF5 expression in a wide variety of neoplasms and that interference with ATF5 function selectively triggers death of breast carcinoma cells. Such findings may have potential therapeutic application.

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic alpha1C and beta1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC50=340 nM) and 5-HT (EC50=81 nM) which was completely inhibited by the cAMP antagonist 2',5'-dideoxyadenosine (10 microM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC50=420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20+/-12 and 35+/-5% respectively) in serum-free medium whereas gefitinib (1 nM-10 microM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

Hypoxic Regulation of Id-1 and Activation of the Unfolded Protein Response Are Aberrant in Neuroblastoma

The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation.

Activation of the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) in Aggressive B-Cell Lymphomas.

BACKGROUND The UPR is a prosurvival pathway activated in cells under ER stress induced by the accumulation of unfolded proteins. UPR activation in B cells normally occurs during the differentiation to antibody secreting plasma cells and requires XBP1activation. XBP-1 is a member of the TREB family of transcription factors that exists in the endoplasmic reticulum (ER) as a 33kDa protein, and in the nucleus as an active 50kDa transcription factor. The UPR stimulates two different ER proteins, ATF-6 and Ire-1, to increase XBP-1 transcription and XBP-1 mRNA splicing resulting in the accumulation of the active 50kDa nuclear protein. Moreover XBP1 is a target of proteosome inhibitors and is related to the aggressive behaviour of some carcinomas. The role of the activation of XBP-1 in lymphomas is still unknown.DESIGN: Reactive lymphoid tissues and 25 neoplastic human B-cell lines representing different stages of B-cell development were studied for XBP-1 expression by western blot and XBP-1, PAX-5, Blimp-1/prdm1, MUM-1/IRF-4 and ICSBP1/IRF-8 by immunohistochemistry. XBP-1 activation was assessed in 225 B-cell lymphomas from the archives of the laboratory of pathology by western blot, RT-PCR and immunohistochemistry . To further evaluate whether XBP-1 activation was related to the plasmacytic program or to ER stress signals we analyzed the cell lines by Western blot for XBP-1 and ATF-6 expression.RESULTS We characterize XBP-1 expression in reactive lymphoid tissues, 25 human cell lines and 225 B-cell tumors. In nearly all tonsillar lymphoid cells XBP-1 was detected as a cytoplasmic protein with a paranuclear dot pattern. Nuclear positivity was observed only in scattered centrocytes in the light zone of the germinal centers and in plasma cells, always coexpressed with plasma cell related transcription factors as MUM-1/IRF-4 and Blimp1/prdm1. Active p50XBP-1 was found in 24/25 cell lines by western blot regardless ATF-6 expression and confirmed by immunohistochemistry . Moreover p50XBP1 was found in 27/31(87%) plasmacytomas, 36/64(56%) DLBCL-ABC and in 3/10(30%) DLBCL-GCB and 22/43(51%) plasmablastic lymphomas. Intriguingly, p50XBP1 was detected also in 2/11(18%)BL and 4/25(16%)MCL with blastic features.CONCLUSIONS.XBP-1 is activated in a subset of follicular centre cells committed to plasma cell differentiation and in plasma cells.UPR prosurvival pathways in the neoplastic cell lines are activated independently of the extent of the ATF-6 activation.p50XBP1 is mostly activated in aggressive B-cell lymphomas regardless to the plasmacytic differentiation of the tumours.Thus, p50XBP-1 may be a new molecular target in the treatment of aggressive B-cell malignancies.

Up-Regulation of Dicer, a Component of the MicroRNA Machinery, in Prostate Adenocarcinoma

MicroRNAs are small noncoding 18- to 24-nt RNAs that are predicted to regulate expression of as many as 30% of protein-encoding genes. In prostate adenocarcinoma, 39 microRNAs are up-regulated, and six microRNAs are down-regulated. Production and function of microRNA requires coordinated processing by proteins of the microRNA machinery. Dicer, an RNase III endonuclease, is an essential component of the microRNA machinery. From a gene array analysis of 16 normal prostate tissue samples, 64 organ-confined, and four metastatic prostate adenocarcinomas, we identified an up-regulation of major components of the microRNA machinery, including Dicer, in metastatic prostate adenocarcinoma. Immunohistochemical studies on a tissue microarray consisting of 232 prostate specimens confirmed up-regulation of Dicer in prostatic intraepithelial neoplasia and in 81% of prostate adenocarcinoma. The increased Dicer level in prostate adenocarcinoma correlated with clinical stage, lymph node status, and Gleason score. Western blot analysis of benign and neoplastic prostate cell lines further confirmed Dicer up-regulation in prostate adenocarcinoma. Dicer up-regulation may explain an almost global increase of microRNA expression in prostate adenocarcinoma. The presence of up-regulated microRNA machinery may predict the susceptibility of prostate adenocarcinoma to RNA interference-based therapy.