Variant Cell Lines Research Articles

Abstract 5121: Laminin 511 on human gastric carcinoma cells is a ligand for hepatic asialoglycoprotein receptor involved in liver metastasis

Abstract – Background and Aim – Gastric cancer often metastasizes to the liver and galactose-type lectins were previously proposed to play an important role in hepatic metastases because intravenous administration of galactose at the time of surgical removal of gastric cancer was reported to extend the survival of the patients. Asialoglycoprotein receptor (Asgpr) is a galactose-type calcium-type lectin and a primary candidate molecule involved in this process. Variant cell lines of human gastric carcinoma, AZ521 cells, were obtained by sorting for high and low levels of cell surface bindings of recombinant Asgpr and designated as AZhigh and AZlow cells, respectively. The present report is to assess the metastatic potential of these cell lines and determine the ligand for Asgpr on the surfaces of these gastric carcinoma cells. – Materials and Methods – AZhigh and AZlow cells were injected into nude mice via portal vein and metastatic growth in the liver were evaluated 14 days later. AZhigh and AZlow cells were biotinylated at the cell surfaces and the lysates were prepared. Components bound to immobilized Asgpr were collected and analyzed by SDS-PAGE then differentially expressed components were subjected to MALDI-TOF MS analysis after trypsin digestion. Specific monoclonal antibodies specific for particular components such as laminin α5 chain were used to confirm the identified proteins from the carcinoma cells. – Results and Discussion – Metastatic growth in the liver was more prominent with AZhigh cells than AZlow cells. AZhigh cells adhered to immobilized Asgpr more rapidly than AZlow cells. From components eluted from immobilized Asgpr, laminin α5, β1, and γ1 chains were identified. By flow cytometric analysis, anti-human laminin α5 antibody bound to AZhigh cells but the binding to AZlow were negligible. Similar difference was observed by immunoprecipitation. Laminin α5 chain was also in the culture supernatants of AZhigh cells, but not those of AZlow cells. These results suggest that laminin 511 differentially expressed between AZhigh and AZlow cells and acted as the ligands for Asgpr. Further study focusing on laminin 511 will be needed for elucidating the mechanism of hepatic metastases of gastric cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5121.

Abstract 4412: In vitro activity of the novel tubulin active agent BAL27862 in MDR1(+) and MDR1(−) human breast and ovarian cancer variants selected for resistance to taxanes

Abstract BAL27862 is a synthetic small molecule that induces apoptosis due to the inhibition of tubulin polymerization via a potentially new mechanism of action. The activity of this novel compound was evaluated in human breast and ovarian cancer cell line variants selected for resistance by continuous exposure to taxanes. BAL27862 retained activity against MDR1 (ABCB1)/P-glycoprotein-mediated resistance variants, unlike vinca alkaloids and taxanes. Activity was also observed in the taxane-resistant ovarian 1A9/TxTP50 tumor cell line, which overexpresses TUBB3 and was approximately 35x resistant to taxanes. Moreover, BAL27862 retained activity in ovarian cancer cells with acquired mutations in the class I (M40) β-tubulin isotype (1A9/PTX10, Phe270->Val; 1A9/PTX22, Ala364->Thr) which confer taxane resistance. The MCF-7/TxTP50 breast tumor variant was 9x resistant to taxanes, and 5x resistant to epothilone B, yet retained sensitivity to BAL27862, the vinca alkaloids (vinblastine and vincristine), colchicine and anthracyclines. These cells have significantly reduced levels of the taxane-binding target, the class I β-tubulin isotype, and reduced levels of total α- and β-tubulin relative to wild-type cells, alterations which may be responsible for a 34% reduction in bound BODIPY-paclitaxel as detected by FACS analysis. Evidence of resistance to BAL27862 (3x) was only detected in the ovarian OVCAR-3/TxTP5 variant, which was also resistant to taxanes (8x), the vinca alkaloids (4x) and colchicine (2x) and ixabepilone (2x). Finally, silencing TUBB3 using a specific siRNA sensitized cells to taxanes but did not affect BAL27862 activity in both wild-type MCF-7 and OVCAR-3 cells relative to appropriate controls, suggesting that TUBB3 status may not influence BAL27862 response. Thus, BAL27862 is a new agent with a distinct in vitro activity profile against a broad panel of human breast and ovarian tumor cell lines resistant to standard tubulin-interacting agents. These data strongly support further development of BAL27862 as a novel anticancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4412.

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

NMDA receptors are expressed by small-cell lung cancer and are potential targets for effective treatment

We previously showed that functional N-methyl-D-aspartate (NMDA) receptors are expressed by human neuroblastoma cells. In this study we demonstrate functional NMDAR1 and NMDAR2 receptors are expressed by small-cell lung cancer (SCLC) classical cell lines NCI H146, NCI H345, and DMS 53, by variant cell line NCI H82, and by most SCLC tumors, and that these receptors are important for the growth of human SCLC tumor xenografts in mice. Reverse transcription-polymerase chain reaction demonstrated mRNA for both receptors, with sequences identical to those for human mRNAs, are expressed in all four cell lines, and these generated proteins of the expected sizes 120 and 170 kDa. Cell viability tests showed cell growth was significantly (P < 0.0001) impaired by NMDAR1 antagonists MK-801 and memantine. Ifenprodil and Ro25-6981, NMDAR2B antagonists at the polyamine site, also significantly (P < 0.001) inhibited the growth/survival of these cells. Alternatively, the glycine-binding antagonist, L701, 324, increased viability to 140% and 120% in NCI H345 and NCI H82 cells after 48 hours of incubation. Immunohistochemistry of SCLC tumors with our polyclonal antibodies gave specific positive staining for the NMDAR1 receptor in 8 of 10 tissues examined. Small amounts of these same antibodies significantly reduced the growth of NCI-H345 cells up to 25% (P < 0.001). When NCI H345 cells were grown as tumor xenografts in mice, the growth of these tumors was reduced by 60% (P < 0.001) by treatments with MK-801 over five days. All of these data point to active NMDAR receptors possibly having an important influence on SCLC growth and survival.

Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis

Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

Mechanisms of the formation of radiation-induced chromosomal aberrations

Although much is now known about the mechanisms of radiation-induction of DNA double-strand breaks (DSB), there is less known about the conversion of DSB into chromosomal aberrations. In particular the induction and ‘rejoining’ of chromatid breaks has been a controversial topic for many years. However, its importance becomes clear in the light of the wide variation in the chromatid break response of human peripheral blood lymphocytes from different individuals when exposed to ionizing radiation, and the elevation of the frequency of radiation-induced chromatid breaks in stimulated peripheral blood lymphocytes of around 40% of breast cancer cases. A common assumption has been that chromatid breaks are merely expansions of initiating DSB, although the classic ‘breakage-first’ hypothesis (Sax, Ref. 44) was already challenged in the 50's by Revell [30] who maintained that chromatid breaks were formed as a result of an incomplete exchange process initiated by two interacting lesions of an unspecified nature. Here we argue that both these models of chromatid break formation are flawed and we suggest an alternative hypothesis, namely that a radiation-induced DSB initiates an indirect mechanism leading to a chromatid break. This mechanism we suggest involves the nuclear enzyme topoisomerase IIα and we present evidence from topoisomerase IIα expression variant human cell lines and from siRNA treatment of human cells that supports this hypothesis.

Anticancer activity and mode of action of titanocene C

Titanocenes constitute a class of metal-based anticancer agents that seem to display a mode of action distinct from that of platinum complexes and to be more tolerable with a differing spectrum of activity. In the present study, titanocene C (bis-(N,N-dimethylamino-2(N-methylpyrrolyl)-methyl-cyclopentadienyl) titanium(IV) dichloride) was shown to exhibit antiproliferative activity against human tumor cell lines with a mean IC₅₀ value of 48.3 ± 32.5 µM. In particular, high activity was found against small cell lung cancer (SCLC) cell lines with a profile different from cisplatin. Titanocene C induced cell cycle arrest at the G1/0-S interphase. Cross-resistance to either cisplatin or oxoplatin, respectively, was low for titanocene C and absent for titanocene Y in variant HL-60 cell lines. Alterations in gene expression of NCI-H526 SCLC cells induced by titanocene C were investigated using genome-wide expression arrays. Downregulation was found for genes coding for topoisomerases I and IIα, histones of the HIST1H4 cluster, enzymes involved in glycolysis, components of the cytoskeleton and vesicular transport, among others. In contrast, expression of genes involved in apoptosis, stress response, particularly members of the metallothionein gene cluster 1, DNA damage and growth factors was upregulated following exposure to titanocene C. Approximately 50% of those genes downregulated by titanocene C and cisplatin were concordant, including the previously identified markers of cisplatin-sensitivity, tubulin and stathmin, indicating partial overlap of the pathways affected by these metal complexes. The present findings point helicases/topoisomerases and HIST1H4 core histones out as targets of titanocene C and metallothioneins as putative main effectors of drug resistance.

The Role of Dragon (Repulsive Guidance Molecule-B, RGM-B) in Human Breast Cancer.

Abstract Introduction: Repulsive guidance molecules (RGMs) are involved in embryonic development and iron homeostasis. RGM-A mediates repulsive axonal guidance and neural tube closure, and RGM-C is mutated in juvenile hemochromatosis. RGM-B, also known as Dragon, is a myelin-derived inhibitor of axon growth in the central nervous system. The RGM family was also identified as co-receptors of bone morphogenetic proteins (BMPs), a group of proteins that are involved in development of bones, the differentiation and progression of cancer. However, the role of RGMs played in breast cancer remains unclear. In the present study, we examined the pattern of expression of the RGM family in human breast cancer cells and investigated the impact of Dragon (RGM-B) on BMP-induced cell function in breast cancer cells.Material and Methods: Conventional RT-PCR was performed to screen the expression of RGMs in human breast cancer samples and a range of breast cancer cell lines. Dragon/RGM-B ribozyme transgenes were generated and in order to knock down the Dragon transcript. Subsequently, MDA-231RGMB-Knock-down variants were created by way of the transgenes. A series of cell function assays were employed to investigate any biological effects upon RGM-B knockdown on the breast cancer cells as well as cell's response to recombinant BMP proteins.Results: RGM-A and RGM-C transcripts were barely detectable in breast cancer cell lines (MDA-MB-231 and MCF-7) and tissues. However RGM-B transcripts were expressed in both cell lines. Using anti-RGM-B transgenes, MDA-231RGMB-Knock-down variant cell lines, in which RGM-B transcripts were knocked down, were created. Compared with wild type and control transfection, MDA-231RGMB-Knock-down variants displayed a significant increase in both the adhesiveness and cells growth (p &amp;lt; 0.05, vs the respective controls, for adheresion and for cell growth). Interestingly, RGM-B knockdown did not have any significant effects on the invasiveness of the cells. Finally, wild type breast cancer cells and the RGMB knock down variant cell lines showed a similar response to the treatment with rhBMP-11, indicating that RGM-B is less critical in BMP-11 mediated cell functions.Conclusion: The present study is the first to examine the role of RGM-B in breast cancer and has demonstrated that knock-down of RGMB could enhance breast cancer cells' ability to grow and attach, indicating that RGM-B may act as an inhibitor in breast cancer. This property is unique to RGMB, as this ability is not associated with any other member in the RGM family. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6158.

Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.

Actin in human colon adenocarcinoma cells with different metastatic potential.

Four human colon adenocarcinoma cell line variants with different metastatic potential were used to examine whether a correlation exists between actin level, state of actin polymerization and invasiveness of tumour cells. Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolic fraction of these cells. A statistically significant decrease in G actin level and increase in the state of actin polymerization (measured by F:G actin ratio) were found in the cytosol of three cell variants with higher metastatic potential and invasiveness (EB3, 3LNLN, 5W) compared with the parental cell line (LS180). Our experimental data lead to the conclusion that there is a correlation between the metastatic capacity of human colon adenocarcinoma cells and the state of actin polymerization.

Biological and Molecular Characterization by Integrative Genomics Approach of CMA-03/06, a Newly Established Interleukin-6 Independent Variant of the CMA-03 Human Myeloma Cell Line.

Abstract 1814Poster Board I-840 BackgroundThe growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. ResultsUnlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. ConclusionsOur data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. DisclosuresNo relevant conflicts of interest to declare.

Pharmacogenetic characterization of indacaterol, a novel β2‐adrenoceptor agonist

Indacaterol is a novel beta(2)-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human beta(2)-adrenoceptor and in human primary airway smooth muscle (ASM) cells. Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human beta(2)-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3',5'-cyclic-monophosphate production was used as an outcome measure. In both the low- and high-expression recombinant GEVT 'wild type' cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the beta(2)-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3',5'-cyclic-monophosphate in response to beta(2)-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol. These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of beta(2)-adrenoceptors.

Maspin: a new potential osteosarcoma metastasis suppressor gene.

Objective To screen osteosarcoma metastasis associated genes through integrative anal-ysis of multidimensional genomic data and preliminarily probe the relationship between the genes and os-teosareoma metastasis. Methods A new approach was taken to screen osteosarcoma metastasis associated genes by integrative analysis of DNA copy number alterations in osteosarcoma specimen and expression pro-filing discrepancy in osteosarcoma cell line variants with different metastasis potentials. The expression of the genes in various osteosarcoma cell lines, normal osteoblast cell, osteosarcom specimen and normal bone specimen was detected by RT-PCR, Western blot and immunohistochemisty. The potential role of the genes in clinical prognosis was also analyzed through prospectively reviewing the clinical data of 27 osteosarcom patients. Results The expression of Maspin was found obviously down regulated in MG63-A1 subline with high metastasis potential after mining the gene array data which demonstrated the expression level of 142 genes varied more than 4-fold between MG63 and MG63-AI. Maspin locates at chromosome 18q which was frequently underwent DNA copy number alterations and Loss of heterozygosity according to GEO database. The expression in MG63 and MG63-A1 was proved by RT-PCR and Western blot at mRNA and protein level while it was found highly expressed in normal osteoblast cell and loss of expression in SAOS2 and 143B. Immunohistochemisty analysis revealed that the expression was mild to moderate positive in 9 of 27 osteosar-coma specimen and intense positive in normal bone specimen. Kaplan-Meier analysis identified that the ex-pression was correlated with the prognosis (P=0.032). Conclusion The expression of Maspin is reduced with the increased ability of the metastasis in various osteosarcoma cell lines and correlated with the progno-sis of the osteosareoma patients. These findings indicate that Maspin probably is a potential target for pre-venting osteosarcoma metastasis. Key words: Osteosareoma; Neoplasm metastasis; Genes

Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V

BackgroundA unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.ResultsUsing immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied.ConclusionOur comparative analysis provides further evidence for similarities and differences between three hESC lines in self-renewal, and spontaneous and directed differentiation. These differences may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture.

Editorial Comment on: A Randomized Phase 1 Study of Testosterone Replacement for Patients with Low-Risk Castration-Resistant Prostate Cancer

[2] Loblaw DA, Virgo KS, Nam R, et al. Initial hormonal management of androgen-sensitive metastatic, recurrent, or progressive prostate cancer: 2006 update of an American Society of Clinical Oncology practice guideline. J Clin Oncol 2007;25:1596–605. [3] Tannock IF, de Wit R, Berry WR, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004;351:1502–12. [4] Gomella LG. Contemporary use of hormonal therapy in prostate cancer: managing complications and addressing quality-of-life issues. BJU Int 2007;99(Suppl 1):25–9, discussion 30. [5] Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 1995;9:401–6. [6] Koivisto P, Kononen J, Palmberg C, et al. Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res 1997;57:314–9. [7] Whitacre DC, Chauhan S, Davis T, Gordon D, Cress AE, Miesfeld RL. Androgen induction of in vitro prostate cell differentiation. Cell Growth Differ 2002;13:1–11. [8] Joly-Pharaboz MO, Ruffion A, Roch A, et al. Inhibition of growth and induction of apoptosis by androgens of a variant of LNCaP cell line. J Steroid Biochem Mol Biol 2000;73:237–49. [9] Chuu CP, Hiipakka RA, Fukuchi J, Kokontis JM, Liao S. Androgen causes growth suppression and reversion of androgen-independent prostate cancer xenografts to an androgen-stimulated phenotype in athymic mice. Cancer Res 2005;65:2082–4. [10] Prout Jr GR, Brewer WR. Response of men with advanced prostatic carcinoma to exogenous administration of testosterone. Cancer 1967;20:1871–8. [11] Fowler Jr JE, Whitmore Jr WF. The response of metastatic adenocarcinoma of the prostate to exogenous testosterone. J Urol 1981; 126:372–5. [12] Fowler Jr JE, Whitmore Jr WF. Considerations for the use of testosterone with systemic chemotherapy in prostatic cancer. Cancer 1982;49:1373–7.

Suppression of topoisomerase IIα expression and function in human cells decreases chromosomal radiosensitivity

The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear (‘rejoin’) as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIα. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIα expression. Here we report that suppression of topoisomerase IIα in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIα in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.

Curcumin as a Possible Lead Compound against Hormone‐Independent, Multidrug‐Resistant Breast Cancer

We examine the possible evidence that the phytochemical curcumin may overcome resistance to hormonal and cytotoxic agents in breast cancer. We present our observations on MCF-7R, a multidrug-resistant (MDR) variant of the MCF-7 breast cancer cell line. In contrast to MCF-7, MCF-7R lacks aromatase and estrogen receptor alpha (ERalpha) and overexpresses the multidrug transporter ABCB1 and the products of different genes implicated in cell proliferation and survival, like c-IAP-1, NAIP, survivin, and COX-2. Nevertheless, in cytotoxicity and cell death induction assays, we found that the antitumor activity of curcumin is substantial both in MCF-7 and in MCF-7R. We elaborated the diketone system of curcumin into different analogues; the benzyloxime and the isoxazole and pyrazole heterocycles showed remarkable increases in the antitumor potency both in the parental and in the MDR MCF-7 cells. Furthermore, curcumin or, more potently, the isoxazole analogue, produced early reductions in the amounts of relevant gene transcripts that were diverse (i.e., they were relative to Bcl-2 and Bcl-X(L) in MCF-7 and the inhibitory of apoptosis proteins and COX-2 in MCF-7R) in the two cell lines. Thus, the two compounds exhibited the remarkable property of being able to modify their molecular activities according to the distinct characteristics of the parental and MDR cells. We discuss also how curcumin may (1) exert antitumor effects in breast cancer through ER-dependent and ER-independent mechanisms; and (2) act as a drug transporter-mediated MDR reversal agent. Overall, the structure of curcumin may represent the basis for the development of new, effective anticancer agents in hormone-independent MDR breast cancer.

Isolation and characterization of a highly metastatic hybrid cell line generated between estrogen receptor (ER) negative and ER positive breast cancer cells in mouse bone marrow.

Abstract Abstract #2053 Background: Breast cancer cells in a patient are heterogeneous. How the presence of these heterogeneous cancer cells may contribute to cancer progression is not clear. Experimental evidence indicates that soluble factors produced by aggressive cancer cells may generate metastasis niches for less aggressive cancer cells. In this study, we sought to determine how a growing orthotopic tumor formed by an aggressive ER negative breast cancer cell line might affect the metastatic potential of a less aggressive ER positive breast cancer cell line.&amp;#x2028; Methods and Results: Five-week-old female nude mice were injected orthotopically with metastasis-competent ER negative human breast cancer MDA-MB-231/GFP/Neo cells. After three weeks, metastasis-incompetent human breast cancer ER positive ZR-75-1/GFP/puro cells were inoculated into these mice via intra-cardiac (IC) route and puromycin resistant metastatic ZR-75-1/GFP/puro cells were flushed out of the bone marrow of one mouse and established as a variant cell line called B6. B6 cells have both estrogen-dependent and -independent cells when they were orthotopically injected into female nude mice with or without estrogen supplementation. The cells isolated from an estrogen-dependent tumor were called B6TE and the cells isolated from an estrogen-independent tumor were called B6TC. Further characterization revealed that B6TC cell was more tumorigenic without estrogen supplementation and has a unique metastatic property. More interestingly, B6TC cell was resistant to both puromycin and G418 suggesting it was derived from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. When compared with parental MDA-MB-231/GFP/Neo, the B6TC cells were found to be more metastatic to lung and bone after IC inoculation as detected by GFP imaging, with significant development of paraplegia in the hind limbs after four weeks. More significantly, B6TC mice also developed metastases to brain, which was not observed in the MDA-MB-231/GFP/Neo group. B6TC cell has a low expression of ER alpha and CD24, and high expression of p-ERK, CD44, ALDH. It also expresses vimentin, CXCR4 and Integrin-β1. Its growth is refractory to the anti-estrogens, raloxifene and tamoxifen.&amp;#x2028; Conclusion: Our study indicates that B6 cells generated in the bone marrow were a heterogeneous population of ER positive and ER negative cells with marked differences in their proliferative potential and malignancy. The low ER-expressing B6TC is a novel hybrid cell line generated spontaneously in a metastatic site, which has propensity to metastasize to brain in addition to lung and bone. The expression of CD44 and ALDH indicates that it has stem cell-like features. Further characterization and molecular profiling is underway to reveal the novel biomarkers. This cell line should be a useful model for the investigation of the molecular mechanism of brain metastasis and the therapeutic strategies for the treatment of metastatic breast cancer resistant to anti-estrogens. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2053.

Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2-DE and MS led to the generation of their 2-DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2-DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.