Respective Parental Lines Research Articles

Cell Surface Tetraspanin CD9 Mediates Chemoresistance in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with extremely high mortality due to the appearance of widespread metastases early in its clinical course and rapid acquisition of chemoresistance after initial therapy. A theory of cell adhesion-mediated drug resistance is thought to be a principal mechanism in which extracellular matrix proteins provide a survival advantage against cytotoxic drug-induced apoptosis. We found that the tetraspanin family member CD9 was expressed preferentially in SCLC tumors and metastases from three of seven relapsed patients, whereas chemonaïve primary tumors from 16 patients were CD9 negative with only one exception. Additionally, CD9 was highly expressed on SCLC cell lines rendered resistant to cisplatin or etoposide, and was upregulated in parental chemosensitive cells within 48 hours after exposure to either of these compounds. CD9-expressing chemoresistant SCLC cells adhered more tightly to fibronectin via β1 integrin, but they were less motile than the respective chemosensitive parental lines. Notably, treatment of the chemoresistant cells with chemokine CXCL12 downregulated CD9 and transiently restored motility. Moreover, selective targeting of CD9 by treatment with specific monoclonal antibody ALB6 or a small interfering RNA triggered apoptosis in the chemoresistant cells. Taken together, our findings implicate CD9 in the cell adhesion-mediated drug resistance mechanism, highlighting CD9 as an attractive therapeutic target to improve therapeutic outcomes in SCLC.

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production. The long-term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re-transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational.

An Investigation into a Panel of Isogenic Taxane Resistant Breast Cancer Cell Lines Using Transcriptomic and Genomic Methods.

Abstract Background: Taxanes are a powerful chemotherapeutics used to treat breast cancer. Taxane resistance has become increasingly more prevalent in the clinic; This problem is exacerbated by a lack of understanding of mechanisms underlying taxane resistance.Materials and Methods: 3 isogenic breast cancer cell-line panels: paclitaxel resistant (PACR) MDA-MB-231s/ZR75-1s and docetaxel resistant (DOCR) ZR75-1s were derived by culturing cells in incrementally escalating doses of taxanes over time. MDA-MB-231: native, 5nM, 25nM and 100nM PACR cells, ZR75-1: native, and 5nM, 25nM, and 50nM PACR and ZR75-1: native, and 5nM, 25nM and 50nM DOCR cells were analysed. RNA from native and PACR MDA-MB-231 cell lines was analysed on the Illumina® human ref.8V2 chip. Data were analysed using IlluminaGui. For aCGH, DNA was extracted from MDA-MB-231 PACR, ZR75-1 PACR and DOCR and respective parental lines, labelled and hybridised to a tiling path BAC microarray (resolution ∼50kb). Each cell-line was tested against reference samples of DNA from pooled female blood. Taxane resistant cells were tested against their parental cell line.Results: Illumina data demonstrated that resistance to increasing doses of paclitaxel is associated with sequential increases in mRNA dysregulation in MDA-MB-231 cells.A number of comparisons between the illumina data of native and the PACR cells were made using a p value of P≤0.001. The first comparison made using the illumina data, was between the 5nM PACR cells and the native cell lines: 25 significantly up or downregulated genes were identified. When the 25nM PACR cells were compared to the native cell lines, 225 genes were found to be significantly up or downregulated. Finally, when 100nM PACR cells were compared to native cells, 425 genes were found to be significantly up or downregulated. A group of genes identified that were common to multiple paclitaxel resistant groups included candidates such as YY1, AURKA and CCND2. aCGH has identified significant differences in patterns of copy number loss, gain and amplification in the taxane resistant cell lines, including chromosome loss of 6p and gain of 2p. Illumina and aCGH data were overlaid using specially designed algorithms.Discussion: Both aCGH and expression arrays highlight a progressive acquisition of molecular and transcriptomic alterations in isogenic breast cancer cell lines resistant to increasing doses of taxanes. Further mapping of transcriptomic and genomic profiles will enable identification of key molecular targets for restoration of taxane sensitivity. Genes upregulated in taxane resistant clones identified in the illumina experiment, in particular those whose upregulation is underpinned by DNA copy number gains in resistant cells, may represent genes which may be targeted with inhibitors to test potential candidates for reversing taxane resistance. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1134.

Abstract C2: Reactivation of embryonic stem cell-like program in human cancer cells by diverse manners

Abstract Embryonic stem cell (ESC)-like gene expression signature has been identified in multiple types of cancers and associated with high grade tumors and poor prognosis. The ESC signature is further linked with cancer stem/tumor initiating cells. However, it is unclear whether the ES signature reflects inheritance of a stem cell-of-origin or reactivation during the course of tumor progression. One recent study implicates the latter because c-Myc, but not the other tested oncogenes in the report, is sufficient to reactivate the ESC signature in normal and cancer cells. We aim to induce aggressive cancer phenotype in two well-differentiated human cancer cell lines, A549 of lung adenocarcinoma origin, and MCF-7 of breast cancer origin. In A549 cells, we generated two variants in which one was transduced with a backbone retroviral vector (A549neo) and the other was a clone overexpressing histone deacetylase 6 (HDAC6), a mediator of TGF-β1-induced EMT, upon retroviral transduction (A549HD6). In MCF-7 cells, we established a clone that acquired resistance to TNF-α-induced apoptosis upon chronic exposure (MCF-7TNR). We observed striking similarity between A549HD6 and MCF-7TNR whose appearance was drastically different from their respective parental lines. The appearance of A549HD6 and MCF-7TNR culture resembled ESC culture which features compact cell colonies and high nucleus/cytoplasm ratio. A549HD6 and MCF-7TNR exhibited alteration in cell cycle progression that was highlighted by low percentage of cycling cells in G-1 phase, a signature of ESC. Analysis of gene expression revealed loss of E-cadherin, an epithelial marker. Moreover, profiling stemness and pluropotency markers (TaqMan Human Stem Cell Pluropotency Array, Applied Biosystems) revealed elevated expression of several ESC stemness markers, such as Nanog and UTF1, in A549HD6 and MCF-7TNR. Fourteen out of twenty-two genes that were aberrantly activated in both A549HD6 and MCF-7TNR were targets of Polycomb Repressive Complex 2 (PRC2). Majority of the activated PRC2 targets were either markers of adult stem cells (GATA family members), or promoters or markers of cell types other than the origins of A549HD6 and MCF-7TNR. A549HD6 and MCF-7TNR exhibited greater tumorigenic property in vitro (soft agar assays) and in vivo (xenograft assays) than their counterparts. Furthermore, A549HD6 cell derived tumors displayed disorganized cell mass which was histologcially distinct from A549neo cell derived tumors that features glandular architecture, a signature histology of well-differentiated adenocarcinomas. In summary, we demonstrate reactivation of ESC-like program in well-differentiated human cancer cells. The ESC-like phenotype correlates with greater tumorigenic potential. Because ESC-like reprogramming occurs upon overexpression of EMT mediator or acquisition of apoptosis resistance, our results warrant further investigation of a link between ESC signature and EMT/apoptosis resistance in cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C2.

Bortezomib Resistance in Mantle Cell Lymphoma Is Associated with Expression of a Plasmacytoid Differentiation Program.

Abstract 287Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures:No relevant conflicts of interest to declare.

Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans

Although glyphosate-resistant (GR) technology is used in most countries producing soybeans (Glycine max L.), there are no particular fertilize recommendations for use of this technology, and not much has been reported on the influence of glyphosate on GR soybean nutrient status. An evaluation of different cultivar maturity groups on different soil types, revealed a significant decrease in macro and micronutrients in leaf tissues, and in photosynthetic parameters (chlorophyll, photosynthetic rate, transpiration and stomatal conductance) with glyphosate use (single or sequential application). Irrespective of glyphosate applications, concentrations of shoot macro- and micronutrients were found lower in the near-isogenic GR-cultivars compared to their respective non-GR parental lines Shoot and root dry biomass were reduced by glyphosate with all GR cultivars evaluated in both soils. The lower biomass in GR soybeans compared to their isogenic normal lines probably represents additive effects from the decreased photosynthetic parameters as well as lower availability of nutrients in tissues of the glyphosate treated plants.

Collateral sensitivity to novel thymidylate synthase inhibitors correlates with folate cycle enzymes impairment in cisplatin-resistant human ovarian cancer cells

The cytotoxicity of two novel folate cycle inhibitors with quinoxalinic structure, 3-methyl-7-trifluoromethyl-2(R)-[3,4,5-trimethoxyanilino]-quinoxaline (453R) and 3-piperazinilmethyl-2[4(oxymethyl)-phenoxy]quinoxaline (311S), was tested against a panel of both cisplatin(cDDP)-sensitive and -resistant carcinoma cell lines. Interestingly, the cisplatin-resistant human ovarian line, C13⁎ cells, exhibited collateral sensitivity towards the two compounds when compared to its sensitive parental 2008 cells. In this resistant line, which showed elevated expression of the folate cycle enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), due to cisplatin-resistance phenotype, collateral sensitivity correlated with the greater reduction of enzyme expression. In addition, TS and DHFR expression of the other resistant lines, the human ovarian carcinoma A2780/CP cells and the human breast cancer MDA/CH cells, were decreased in accordance with the similar sensitivity or the low level of cross-resistance to these compounds in comparison to their respective parental lines. Noteworthy, unlike 5-fluorouracil, both drugs reduced the level of TS without inducing ternary complex formation with the co-substrate and the nucleotide analogue. Median effect analysis of the interactive effects of cisplatin with the two quinoxalines mainly showed additive or synergistic cell killing, depending on schedules of drug combinations. In particular, synergistic effects were more often obtained, even on the resistant cells, when cisplatin was added at the beginning of the treatment. These results indicate that, despite the possibility of other mechanisms being involved, inhibition of TS cycle enzymes plays an important role in the pharmacology of these compounds, which might also represent a useful component in drug treatment protocols against cDDP-resistant cells.

Aphid feeding response and microsatellite-based genetic diversity among diploidBrachypodium distachyon(L.) Beauv accessions

False brome grass,Brachypodium distachyon(L.) Beauv, has been proposed as a new model species to bridge rice and temperate cereal crops for genomics research. However, much basic information for this species is still lacking. In this study, six diploidB. distachyon(2n = 2x = 10) accessions (Bd1-1, Bd2-3, Bd3-1, Bd18-1, Bd21 and BD29) were evaluated for their response to infestation by two cereal aphid pests of common wheat (Triticum aestivumL.): the greenbug,Schizaphis graminumRondani, and the Russian wheat aphid (RWA),Diuraphis noxiaMordvilko. Through database mining ofB. distachyonexpressed sequence tag (EST) and genomic DNA sequences, 160 EST- and 21 genomic microsatellite markers were developed and used to evaluate genetic diversity among theB. distachyonaccessions. All six accessions were resistant to RWA biotype RWA1 but showed distinct responses to feeding by greenbug biotypes C and E, as well as RWA2 RWAs. Although microsatellite-based genetic diversity among different accessions was generally low, Bd1-1 and BD29 were the most diverged from the other four lines. The genetic divergence was correlated with geographical distances between theBrachypodiumaccessions. Comparison of simple sequence repeat polymorphisms in three inbred lines (Bd2-3, Bd3-1 and Bd18-1) with their respective original parental lines revealed no effect of inbreeding on genetic diversity. Phylogenetic analysis suggested thatAegilops tauschii(Coss.) Schmal., the D genome donor of common wheat, was closer toB. distachyonthan to rice. The greenbug - B. distachyonsystem seems to be a model of choice for plant–aphid interaction studies in the grass genome.

In vitro growth inhibition of cisplatin-resistant human lung cancer cell lines by recombinant human tumor necrosis factor and/or recombinant human interferon-gamma by virtue of collateral sensitivity.

The colony-inhibitory effects of recombinant human tumor necrosis factor (rH-TNF) and recombinant human interferon-gamma (rH-IFN-gamma) were evaluated in four human lung cancer cell lines and their cisplatin-resistant sublines. The cell lines tested were PC-7 and PC-9 (adenocarcinoma), H69 and N231 (small cell lung cancer) and four cisplatin-resistant sublines, PC-7/1.0, PC-9/0.5, H69/0.2 and N231/0.2, which were 20.0, 7.1, 4.8 and 8.4 fold resistant to cisplatin, respectively, compared to the respective parental cell line in terms of IC50 in a soft agar colony assay. All parental cell lines were resistant to rH-TNF and rH-IFN-gamma, alone or in combination. However, two resistant sublines showed sensitivity to rH-TNF and rH-IFN-gamma. Colony formation by PC-9/0.5 was significantly inhibited, in the absence or presence of cisplatin, by 10(2) U/ml of rH-TNF (less than 50% of control) and the inhibition was synergistic with that produced by 10(3) or 10(4) U/ml of rH-IFN-gamma. RH-IFN-gamma inhibited the colony formation of H69/0.2 only at the highest concentration tested (10(4) U/ml) (less than 50% of control) and the combined effect with rH-TNF was additive. These results suggest that rH-TNF and rH-IFN-gamma may have some potential in overcoming cisplatin resistance by virtue of collateral sensitivity.

SSR Heterogenic Patterns of Maize Parental Lines and Prediction of Hybrid Performance

ABSTRACTTo investigate the potential of molecular markers in predicting hybrid performance, a set of 58 inbred lines were analyzed using 80 SSR primer pairs. The correlation between genetic divergence of inbred lines and the performance of hybrids was analyzed in the three sets of crosses including an 8x8 Diallel, a 6x6 Line x Tester (LxT) and testcross of all the inbreds with two testers. The crosses along with their respective parental lines were individually evaluated on alpha-lattice designs with two replications at two environments. Pair-wise genetic distance (GD) was estimated based on Jaccard, and simple matching (SM) coefficients. Significant correlation was found between GD value of parental lines and hybrid performance for the testcross and diallel data. In diallel analysis significant correlation was observed between total grain yield per ear (TGW) and genetic distance based on SM coefficient, whereas the correlation of GD and specific combining ability of hybrids for this trait was not. Through the stepwise multiple regression analysis a total of 19 informative SSR markers distributed over all chromosomes, except chromosomes 7 and 8, were detected. GD values based on informative markers in general were grater compared to that of based on all markers and significant improvement was observed in the correlations between GD estimates based on informative markers and TGW as well as SCA. Partitioning of genetic distances into general GD (GGD) and specific GD (SGD) revealed a large positive or negative GGD. It was found that GGD accounted for more variation of GD in comparison with SGD. Simple correlations between SGD for different measures and TGW as well as SCA were low and non-significant. This could be probably attributed to non-significant SCA of TGW, besides lower contribution of SGD to total GD variance.

Behavioral and electrophysiological responses of Helicoverpa assulta, H. armigera (Lepidoptera: Noctuidae), their F1 hybrids and backcross progenies to sex pheromone component blends

Two sibling species, Helicoverpa assulta and Helicoverpa armigera both use (Z)-9-hexadecenal and (Z)-11-hexadecenal as their sex pheromone components but in almost reversed ratios, 93:7 and 3:97, respectively. H. assulta and H. armigera males performed upwind flight in response to the H. assulta sex pheromone blend (93:7). H. armigera responded strongly to the H. armigera blend (3:97), whereas H. assulta males remained inactive upon exposure to this blend. Both species gave clear dose-dependent electrophysiological responses to (Z)-11-hexadecenal. However, (Z)-9-hexadecenal evoked strong dose-dependent electrophysiological responses in H. assulta males but not in H. armigera. The two male F1 hybrids exhibited similar behavioral responses to two sex pheromone blends and electrophysiological responses to two pheromone components as H. armigera males. This indicated that H. armigera genes appear dominant in determining the behavioral response and electrophysiological responses. Behavioral and electrophysiological responses of backcrosses of male F1 hybrids (H. armigera female x H. assulta male) with female H. assulta and H. armigera were close to that of H. assulta and H. armigera, respectively. However, backcrosses of female F1 hybrids (H. assulta female x H. armigera male) with male H. assulta and H. armigera showed reduced behavioral responses but normal electrophysiological responses compared to males of the respective parental line.

Heterozygosis drives maize hybrids to select elite 2,4-diacethylphloroglucinol-producing Pseudomonas strains among resident soil populations

By comparing the distribution of two genomic markers among Pseudomonas strains recovered from the rhizosphere of two maize hybrids with those of strains recovered from the rhizosphere of their four respective parental lines, we showed that both hybrids supported more elite probiotic strains than the parents. Elite Pseudomonas strains showed genomic potential for both an appropriate in vitro 2,4-diacetylphloroglucinol (DAPG) productivity, and a superior root-colonization ability. The actual biocontrol and root-colonization abilities of these strains were confirmed by bioassays on five fungal strains and on axenic maize plants. Furthermore, results on the abundance and genetic diversity of resident DAPG+ Pseudomonas strains indicated that each hybrid was able to select its own specific DAPG+ population, whereas the four parental lines were not. The evidence that heterozygosis can drive maize plants to select elite probiotic rhizospheric DAPG+ Pseudomonas strains opens the way to a new strategy in the set up of plant breeding for low-input and organic agriculture.

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.

Increased lysosomal uptake of methotrexate-polyglutamates in two methotrexate-resistant cell lines with distinct mechanisms of resistance

Methotrexate (MTX) resistance in mitoxantrone-selected MCF7/MX cells and in MTX-selected CEM/MTX cells is associated with reduced drug accumulation, albeit caused by different mechanisms. In addition, in both resistant cell lines the proportion of active long-chain MTX-polyglutamate (MTX-PG) metabolites is reduced relative to that in the respective parental cell line. Previous studies by others have implied that increased lysosomal uptake could affect the rate of MTX-PG hydrolysis, and hence the length distribution of the polyglutamate chains. However, in the two cell line pairs studied, the number of lysosomes per cell was not different between the corresponding parental and resistant cells. Instead, we observed a two- to three-fold increased facilitative uptake of MTX-Glu 4 by the lysosomes from these two independently derived MTX-resistant cell lines, compared to uptake by lysosomes from their corresponding parental cells. Enhanced lysosomal uptake of MTX-Glu 4 was reflected in an increased maximal uptake velocity, without a change in the apparent substrate affinity. In addition, the rate of MTX efflux from lysosomes from CEM/MTX cells was two-fold faster than from lysosomes from CEM cells. Consistent with this observation, the relative amount of short-chain MTX-Glu 1+2 species, as a fraction of the total amount of all MTX-Glu 1−4 species combined, was only half as large in lysosomes from CEM/MTX cells as in lysosomes from CEM cells. Together, these results suggest the possibility that increased lysosomal uptake, and hence enhanced sequestration of MTX-PGs in resistant cells, contributes to the development of high-level MTX resistance by decreasing the cytosolic levels of MTX-PGs.

A study of cytotoxic synergy of UCN-01 and flavopiridol in syngeneic pair of cell lines

Flavopiridol and UCN-01 are two novel protein kinase inhibitors with diverse cellular effects that may complement each other with regards to induction of apoptosis. HeLa cells engineered to overexpress human survivin (HeLa-S) were at least approximately 4.8-fold resistant to UCN-01 relative to proliferation observed in control HeLa cells (HeLa-V). Flavopiridol cytotoxicity as measured using the MTT assay was unaffected in HeLa-S cells when compared with HeLa-V cells. Similarly, simultaneous treatment of HeLa-V cells with flavopiridol and UCN-01 for 72 hours did not result in synergistic inhibition of proliferation; however, in HeLa-S cells, this combination resulted in synergistic inhibition of cell proliferation. Flavopiridol and UCN-01 augmented apoptosis in HeLa-S cells (as compared with HeLa-V cells) as measured by caspase-3 cellular activity assay, DNA fragmentation and PARP cleavage by western blot. In HeLa-V and -S cells, combination treatment resulted in caspase-8 cleavage. Caspase-9 was expressed in HeLa-V cells; however, there was a marked reduction of caspase-9 content in HeLa-S cells only. Combination treatment resulted in a significant reduction in survivin abundance in HeLa-S and SKBR3-UR cells, but not in their respective parental lines. The synergy of Flavopiridol and UCN-01 are selectively toxic to survivin-overexpressing cell lines and the mechanism of toxicity involves caspase-dependent cell death.

Correlation of Cs/foreign gas diffuse bands with theoretical interaction potentials

Fluorescence excitation is employed to survey diffuse spectral bands in mixtures of Cs with He, Ne, Ar, Kr, Xe, or N 2 over the wavelength range 430–470 nm. Direct fluorescence and sensitized fluorescence at low pressures are also utilized to associate various diffuse bands with their respective atomic parent lines. The line-band associations, along with the positions and strengths of the bands, are then correlated with theoretical potentials for the collision pairs. The potentials exhibit a rich structure in the excited state which qualitatively describes many of the diffuse band characteristics and lead to the following conclusions: (1) red satellite bands are correlated with long-range (20–30 a.u.) shallow potential wells in the excited state, (2) violet satellite bands are correlated with maxima in the excited state potentials in the range 10–20 a.u., and (3) detached bands are formed near avoided crossings in the range 6–10 a.u. A diagnostic technique for foreign gas density is also demonstrated for N 2 and He.

Molecular characterization of the Smyth chicken sublines and their parental controls by RFLP and DNA fingerprint analysis

The Smyth line (SL) chicken, a model for autoimmune human vitiligo, is characterized by a spontaneous posthatch epidermal pigment loss (vitiligo). Even though the immunological and morphological changes accompanying the vitiligo process have been well studied, the genetics of this phenomenon remains elusive. The SL lines have been maintained by nonpedigreed matings since their inception, and therefore, the inbreeding status is unknown. The present study was designed to provide an estimate of the inbreeding coefficients and the molecular genetic profiles of the SL sublines, each homozygous for a different MHC haplotype and their MHC-matched parental control (BL) sublines. The DNA fingerprint analysis revealed that there is a moderate level of inbreeding within the SL and BL parental sublines. Of the two SL sublines studied, SL101 had the highest level of inbreeding (0.948). Similarly, its parental control line (BL101) was more inbred than the parental subline of SL102 (BL102). The very high level of similarity between the SL sublines and their respective parental control lines is shown further by the similarity index (SI) estimates (SI between SL101 and BL101 was 0.949 and that between SL102 and BL102 was 0.932). Restriction fragment length polymorphism (RFLP) analysis of the endogenous viral genes (avian leukosis virus subgroup E, ALVE) showed that five ALVE-related BamH1 fragments were present in the SL101 and four in SL102 sublines, whereas the parental BL101 and BL102 sublines had five and six fragments, respectively. SL101 and SL102 shared two fragments, but the frequencies were different. Similarly, BL101 and BL102 shared two fragments. SL101 and BL101 shared three fragments, and SL102 and BL102 also shared three fragments.

MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines.

Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were established from the parental prostate cancer cell lines DU145 and PC-3 respectively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 &mgr;g/ml. As cells adapted to each concentration of DOX. the drug concentration was increased by 0.001 &mgr;g/ml. The chemosensitivity of each line was determined by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5-10-fold and 1.3-2.8-fold increase in resistance to anthracyclines, vinblastine (VLB) and mitozantrone (Mito), respectively. Verapamil (5 &mgr;M) partially reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of (3)H-daunorubicin demonstrated a 3 fold decrease in the level of intracellular (3)H-daunorubicin in the PC 0.03 and DU 0.03 resistant lines compared with their respective parental line. This effect was partially reversed by 5 &mgr;M verapamil. The expression of MDR1 and MRP genes was analysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodies J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern blot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 transcripts in any of the prostate cancer cell lines and the three Pgp monoclonal antibodies failed to reveal expression of Pgp. A 2-4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and PC 0.03 lines were demonstrated by both Northern blotting and RT-PCR consistent with the findings observed after staining by the two specific monoclonal antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fold increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regulated at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines. Prostate Cancer and Prostatic Diseases (2000) 3, 66-75

Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes.

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.

Heterosis and developmental stability of body and organ weights at hatch for parental line broiler breeders and specific crosses among them

Body, yolk sac, left and right shanks with toes, empty left and right ceca, left and right lungs, heart, and bursa of Fabricius weights were obtained at hatch for 50 chicks from each of five commercial broiler parental lines (three sire and two dam) and three F1 crosses involving them. Differences among stocks and between sexes were inconsistent among mating combinations. Although correlation coefficients between yolk-free chick weight with organ weights were generally stock specific, they were high (> 0.75) with shank weight, intermediate with heart and lung weights, and low (< 0.25) with ceca and bursa weights. Heart:lung ratios of all F1 crosses were greater than those for their respective parental lines; however, the degree of heterosis differed among populations. Developmental stability, as measured by percentage relative asymmetry, was less in two of the sire parental lines than in their respective dam lines and F1 crosses.