Cancer/testis antigen CSAGE is concurrently expressed with MAGE in chondrosarcoma

Differential display-polymerase chain reaction was used to compare gene expression between human chondrosarcoma cell lines and normal cartilage. A new gene, CSAGE, has been cloned and belongs to a gene family that includes the taxol resistance associated gene (TRAG)-3. CSAGE, like TRAG-3, does not confer resistance to taxol when transfected in vitro. Both genes have alternatively spliced variants. CSAGE and TRAG-3 are expressed in chondrosarcoma, melanoma, and cartilage and testis, but not in other normal tissues. TRAG-3 has been reported to be a cancer/testis antigen. Our results suggest that CSAGE belongs to the growing list of cancer/testis antigens as well. In all of the CSAGE positive samples, the melanoma antigen gene family was also expressed. This is the first report on the expression of cancer/testis antigens in chondrosarcoma.     

Differential partitioning of Gαi1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells

Background

Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results

Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Gαi1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the α-subunit of the G protein have been identified and the Gαi (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Gαi1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Gαi1 in these cells. Moreover, Gαi1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Gαi1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion

Based on the opposing effects of taxol and the Gαi1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Gαi1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.     

Spontaneous CD4+ T cell responses against TRAG-3 in patients with melanoma and breast cancers

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-gamma-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRbeta gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-3(34-48) is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.     

Study of multi-drug resistant mechanisms in a taxol-resistant hepatocellular carcinoma QGY-TR 50 cell line

Cancer chemotherapy with taxol often fails due to acquired resistance of cancer cells, which is frequently associated with an overexpression of P-gp and alterations of beta-tubulin. A taxol-resistant cell line, QGY-TR50, derived from a human hepatocellular carcinoma (HCC) QGY-7703 cell line was used to investigate the mechanisms of taxol-resistance. QGY-TR50 cells showed more than 250-fold resistance to taxol and exhibited cross-resistance to other drugs including actinomycin D, doxorubicin, vinblastine, and vincristine. P-gp was highly expressed in QGY-TR50 cells. Expression levels of five human beta-tubulin isotypes (betaI-, betaII-,betaIII-, betaIva, and betaIvb-tubulin) were examined by real-time semi-quantitative PCR. Comparing with QGY-7703 cells, QGY-TR50 cells did not show any significant change in the expression levels of betaI-, betaIva, and betaIvb-tubulin. While a 1.2-fold increased in betaII-tubulin and a 0.5-fold decreased in betaIII-tubulin levels were observed. All results suggest that the P-glycoprotein could be one key factor involved in enhancing drug resistance in QGY-TR50 cells.     

Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line

Pulse selections on a chemotherapy naive squamous lung carcinoma cell line, SKMES-1, with clinically relevant concentrations of taxanes (taxol or taxotere) resulted in the development of a stable taxotere-resistant variant, SKMES-1-Taxotere and an unstable taxol-resistant variant, SKMES-1-Taxol. Both variants exhibited increased invasiveness in vitro. The unstable nature of SKMES-1-Taxol facilitated looking at factors involved in loss of taxol resistance and increased invasion. The taxotere and taxol-resistant cell lines were 5.9-fold and 12.5-fold resistant to taxotere and taxol respectively. Alterations in expression of/or point mutations in tubulin, the main target of taxanes, is a major mechanism or resistance. However, alterations in expression of beta tubulin were not consistent in the taxane-selected variants. Cross-resistance to adriamycin, vincristine and etoposide (VP-16) was consistent with overexpression of P-glycoprotein (P-gp). However, P-gp alone is not sufficient to confer the complete multiple drug resistance phenotype as all cell lines exhibited cross-resistance to 5-Fluorouracil (5-FU) and more than one mechanism has been linked to taxane resistance. There was no correlation between the fall of taxol resistance in SKMES-1-Taxol and P-gp expression indicating the loss in resistance to be independent of P-gp expression. Furthermore, resistance to the other drugs was not unstable in SKMES-1-Taxol suggesting some parallel mechanisms of resistance. Two-dimensional electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify alterations in expression of specific proteins associated with taxane resistance. A large number of differentially regulated proteins were identified in the resistant and invasive variants affecting cellular processes including stress response, protein turnover and cytoskeleton proteins.     

Isolation of a taxol-resistant Leishmania donovani promastigote mutant that exhibits a multidrug-resistant phenotype

We raised a strain of Leishmania donovani in the laboratory that was resistant to 500 nM taxol. The IC50 of the wild-type strain for taxol was 35 nM and that of the taxol-resistant strain (T-500) was 1 microM. The T-500 strain exhibited a Mdr phenotype; it was also resistant to other unrelated drugs like vinblastine, adriamycin and the commonly used antimonial drugs pentostam and glucantime. Verapamil (20 nM), a calcium channel blocker, was found to reverse the resistance of T-500 to taxol. Acquired resistance to taxol has been reported to be mediated by alterations involving tubulin in cancer cells. Thus polymerisation assays with tubulin fractions in wild-type versus taxol-resistant cells (T-500) were performed in vitro. The tubulin fraction from T-500 was more resistant to in vitro polymerisation than the tubulin isolated from the wild-type, suggesting that this is one means by which the parasite may acquire resistance to taxol.     

Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.     

The nature of the modification at position 37 of tRNAPhe correlates with acquired taxol resistance

Acquired drug resistance is a major obstacle in cancer therapy. Recent studies revealed that reprogramming of tRNA modifications modulates cancer survival in response to chemotherapy. However, dynamic changes in tRNA modification were not elucidated. In this study, comparative analysis of the human cancer cell lines and their taxol resistant strains based on tRNA mapping was performed by using UHPLC–MS/MS. It was observed for the first time in all three cell lines that 4-demethylwyosine (imG-14) substitutes for hydroxywybutosine (OHyW) due to tRNA-wybutosine synthesizing enzyme-2 (TYW2) downregulation and becomes the predominant modification at the 37^th position of tRNA^phe in the taxol-resistant strains. Further analysis indicated that the increase in imG-14 levels is caused by downregulation of TYW2. The time courses of the increase in imG-14 and downregulation of TYW2 are consistent with each other as well as consistent with the time course of the development of taxol-resistance. Knockdown of TYW2 in HeLa cells caused both an accumulation of imG-14 and reduction in taxol potency. Taken together, low expression of TYW2 enzyme promotes the cancer survival and resistance to taxol therapy, implying a novel mechanism for taxol resistance. Reduction of imG-14 deposition offers an underlying rationale to overcome taxol resistance in cancer chemotherapy.     

High expression of class III β-tubulin has no impact on functional cancer cell growth inhibition of a series of key vinblastine analogs

Clinical association studies have implicated high expression of class III β-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III β-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3–10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III β-tubulin unlike the taxanes. Most significant are the results with 4–6, a subset of 20′ amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2–2-fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux.     

Synergistic effect of tumor necrosis factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant human ovarian tumor cell lines

Recent studies have demonstrated that diphtheria toxin (DTX) also mediates target cell lysis, and the mechanism of cytotoxicity has many features similar to those of cytotoxicity mediated by TNF-alpha. Thus, we hypothesized that DTX and TNF-alpha, used in combination, may result in either additive or synergistic cytotoxic activity. This was examined on three human ovarian carcinoma cell lines chosen for their differing sensitivities to TNF-alpha and DTX, i.e., 222, which is sensitive to both TNF-alpha and DTX, 222TR, a TNF-alpha-resistant DTX-sensitive variant of 222, and SKOV-3, which is resistant to both DTX and TNF-alpha. The simultaneous use of DTX and TNF-alpha at suboptimal concentrations resulted in synergistic cytotoxic activity against all three lines tested, thus overcoming the TNF-alpha resistance of 222TR and the double resistance of SKOV-3. DNA fragmentation was observed in all three lines treated with DTX and TNF-alpha and occurred as early as 4 h after treatment. Cycloheximide, actinomycin D, or emetine, at concentrations causing greater than 90% protein synthesis inhibition, did not result in cytotoxicity alone or synergy with TNF-alpha, suggesting that synergy by DTX was not due to its ability to inhibit protein synthesis. The use of energy poisons and pH conditions that inhibit DTX-mediated cytotoxicity resulted in the abrogation of synergy. These findings show that the two cytotoxic agents TNF-alpha and DTX, when used at suboptimal concentrations, synergize in their cytotoxic activity against sensitive and resistant cell lines. Because the SKOV-3 cell line used here is also resistant to chemotherapeutic drugs, combination treatment with DTX and TNF-alpha may be beneficial in overcoming drug resistance.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway

Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.     

Methyl-CpG-DNA binding proteins in human prostate cancer: expression of CXXC sequence containing MBD1 and repression of MBD2 and MeCP2

We analyzed gene expression of MBD1, MBD2, MBD3, MBD4, and MeCP2 and protein expression of MBD1, MBD2, and MeCP2 in prostate cancer cell lines, benign prostate epithelium (BPH-1) cell line, 49 BPH tissues, and 46 prostate cancer tissues. The results of this study demonstrate that MBD2 gene is expressed in all samples and MeCP2 gene is expressed in all cancer cell lines but not in BPH-1 cell line. However, there was no protein expression for MBD2 and MeCP2 in cancer cell lines and cancer tissues. For CXXC sequence containing MBD1, both protein and mRNA were expressed in cancer cell lines, cancer tissues, BPH-1 cell line, and BPH tissues. We observed that, in BPH tissues and low-grade cancer tissues, MBD1 protein expression was very high and gradually decreased with increase of cancer grade. Treatment of cancer cell lines with proteasome inhibitor (MG-132) did not restore expression of MBD2 and MeCP2 proteins. When prostate cancer cell lines were treated with hypomethylating agent, 5-aza-2(')-deoxycytidine (DNMT inhibitor), HDAC1 and HDAC2 expression was decreased. This is the first report demonstrating that CXXC sequence containing MBD1 is overexpressed and can be the major factor of hypermethylated chromatin segments through HDAC1/2 translocation and histone deacetylation in human prostate cancer.     

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro-apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH-EP1 and SH-SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N-terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N-terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH-SY5Y) or lost (SH-EP1) clonogenic survival after taxol treatment. Suppression of taxol-induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH-SY5Y cells. The data suggest that a further drug damage-induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol-resistant cells.     

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.     

Alternative splicing is associated with decreased expression of the redox proto-oncogene thioredoxin-1 in human cancers

Human thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human primary tumors, where it is associated with rapid cell proliferation and inhibited apoptosis. Mutation scanning denaturing high-performance liquid chromatography of Trx-1 mRNA in 58 human tumor cell lines found no evidence for changes in the base sequence of human Trx-1 mRNA. An alternatively spliced form of Trx-1 mRNA lacking exons 2 and 3 was found in 7 of the cell lines but it was not translated. The cell lines having the alternatively spliced Trx-1 mRNA had 73% lower total Trx-1 mRNA than the other cell lines, suggesting that alternative splicing may control the level of Trx-1 mRNA in some cancer cells.