Line S1 Research Articles

An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield

No previous reports have described the effects of an increase in endogenous melatonin levels on plant yield and reproduction. Here, the phenotypes of melatonin-rich transgenic rice plants overexpressing sheep serotonin N-acetyltransferase were investigated under field conditions. Early seedling growth of melatonin-rich transgenic rice was greatly accelerated, with enhanced biomass relative to the wild type (WT). However, flowering was delayed by 1 wk in the transgenic lines compared with the WT. Grain yields of the melatonin-rich transgenic lines were reduced by 33% on average. Other phenotypes also varied among the transgenic lines. For example, the transgenic line S1 exhibited greater height and biomass than the WT, while the S10 transgenic line showed diminished height and an increase in panicle numbers per plant. The expression levels of Oryza sativa homeobox1 (OSH1) and TEOSINTE BRANCHED1 (TB1) genes, two key regulators of meristem initiation and maintenance, were not altered in the transgenic lines. These data demonstrate that an alteration of endogenous melatonin levels leads to pleiotropic effects such as height, biomass, panicle number, flowering time, and grain yield, indicating that melatonin behaves as a signaling molecule in plant growth and reproduction.

A SEARCH FOR C II 158 μm LINE EMISSION IN HCM 6A, A Lyα EMITTER AT z = 6.56

We report a Plateau de Bure interferometer search for CII-158$\mu$m emission from HCM6A, a lensed Lyman-$\alpha$ emitter (LAE) at $z = 6.56$. Our non-detections of CII-158$\mu$m line emission and 1.2mm radio continuum emission yield $3\sigma$ limits of L$_{\rm CII} < 6.4 \times 10^7 \times (\Delta V/100 km s^{-1})^{1/2}$ L$_\odot$ on the CII-158$\mu$m line luminosity and S$_{\rm 1.2mm} < 0.68$ mJy on the 1.2mm flux density. The local conversion factor between L$_{\rm CII}$ and star formation rate (SFR) yields an SFR $< 4.7$ M$_\odot$ yr$^{-1}$, $\approx 2$ times lower than that inferred from the ultraviolet (UV) continuum, suggesting that the local factor may not be applicable in high-$z$ LAEs. The non-detection of 1.2mm continuum emission yields a total SFR $< 28$ M$_\odot$/yr; any obscured star formation is thus within a factor of two of the visible star formation. Our best-fit model to the rest-frame UV/optical spectral energy distribution of HCM6A yields a stellar mass of $1.3 \times 10^9$ M$_\odot$ and an SFR of ~10 M$_\odot$/yr, with negligible dust obscuration. We fortuitously detect CO J=3-2 emission from a $z=0.375$ galaxy in the foreground cluster Abell370, obtaining a CO line luminosity of L$^\prime ({\rm CO}) > (8.95 \pm 0.79) \times 10^8$ K km s$^{-1}$ pc$^2$, and a molecular gas mass of M$({\rm H_2}) > (4.12 \pm 0.36) \times 10^9$ M$_\odot$, for a CO-to-H$_2$ conversion factor of 4.6 M$_\odot$ (K km s$^{-1}$ pc$^2$)$^{-1}$.

Targeting HMGB1 inhibits ovarian cancer growth and metastasis by lentivirus‐mediated RNA interference

High-mobility group box 1 (HMGB1), a nuclear and extracellular protein, is implicated in the development and progression of some types of cancers. However, no information is available to date regarding the function of HMGB1 in ovarian cancer. In this study, we performed cDNA microarray analysis and identified HMGB1 as a gene dramatically elevated in the highly invasive subclone S1 compared with the low invasive subclone S21 derived from the same cell line SKOV3. Then lentivirus vector with HMGB1 shRNA was constructed and infected the highly invasive cell line S1, A1, and HO8910PM. Real-time RT-PCR, Western blot, and IHC results confirmed the down-regulation of HMGB1 expression by its shRNA was about 80-90% at both the mRNA and protein levels. Knockdown of HMGB1 significantly suppressed ovarian cancer cell proliferation and induced cell cycle arrest at the G1/G0 phase, which was accompanied by decreased expressions of cyclin D1 and PCNA. Furthermore, knockdown of HMGB1 induced ovarian cancer cell apoptosis, which was mediated by increased expression of Bax and decreased expression of Bcl-2. Finally, knockdown of HMGB1 significantly inhibited ovarian cancer cell invasion and metastasis, which was regulated by decreased expressions of MMP2 and MMP9. Serum HMGB1 levels in patients with epithelial ovarian cancer were significantly higher than that in patients with benign ovarian tumor and healthy controls. These results indicate that HMGB1 is a newly identified gene associated with ovarian cancer growth and metastasis. HMGB1 may serve as a new therapeutic target for the treatment of ovarian cancer in the future.

Abstract 1533: Sunitinib reverses ABCG2/BCRP-mediated multidrug resistance by inhibiting its efflux activity

Abstract Background and objective: Overexpression of ATP-binding cassette (ABC) transporters, which play a key role in the development of multidrug resistance (MDR), is a significant impediment to successful cancer treatment. Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we investigated in vitro the possible interaction of sunitinib with the major members of the ABC transporters: P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP). Methods: Cytotoxicity tests were performed using the MTT assay; The intracellular accumulation of doxorubicin and rhodamine 123 (Rho123) was analysed by flow cytometry; Membrane vesicles were prepared by the nitrogen cavitation and transport assays were performed using the rapid filtration method to confirm the effect of sunitinib on the transporter activity of ABCG2; Western Blotting was used to determine the protein expression; and reverse transcription-PCR was used to assess the expression of ABCG2 at mRNA level. Results: The IC50 concentration for parental cell line S1 to topotecan or doxorubicin were 0.135 ± 0.056 µM and 0.165 ± 0.009 µM, respectively; However, the concentration of topotecan or doxorubicin required to inhibit ABCG2- overexpressing cell line S1-M1-80 by 50% was about 5.132 ± 2.024 and 6.845 ± 1.035 µM; Coincubation with 2.5 sunitinib, the concentrations required to inhibit the growth of S1-M1-80 cells by 50% for topotecan was 0.091 ± 0.012 µM, and for doxorubicin was 0.181 ± 0.037 µM, respectively. Sunitinib potently reverses ABCG2-mediated resistance to topotecan and doxorubicin in vitro and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1-overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1-overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine-123 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [3H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Conclusions: Sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic. 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 1533.

Simultaneous inhibition of EGFR and c-Met pathways in a panel of NSCLC cell lines

22223 Background: The EGFR inhibitor, erlotinib, prolongs survival in chemotherapy-resistant NSCLC patients, although < 50% respond. Reportedly, simultaneous inhibition of EGFR (by gefitinib) and c-Met (by PHA-665752) was antiproliferative in a line selected for resistance to gefitinib alone. We hypothesized that in lines not previously exposed to either agent, simultaneous inhibition of EGFR and c-Met pathways would slow NSCLC growth more than targeting either single pathway. Methods: NSCLC lines were cultured in 96-well plates at 5000 cells/well. After 24 h, erlotinib (OSI) and the c-Met inhibitor SU11274 (Sigma) were added. Cells were recovered 3–5 d later by trypsinization and counted by hemacytometer. Receptor activation and signaling were studied in A549 by Western blotting following serum deprivation, incubation with inhibitors (3.5 h) and EGF (15 m). Results: In A549, E2 suppressed EGFR phosphorylation (pY1068), and S3 suppressed c-Met pY1230/1234/1235, confirming pathway targeting. Both drugs suppressed phosphorylation of Akt at S473 and Erk1/2 at T202/Y204, consistent with growth inhibition. Conclusions: For all lines except Calu3, S5 was more antiproliferative than E10, demonstrating that c-Met is a target of interest for NSCLC. By the Bliss criterion, the effect of drugs acting independently should equal the product of the effects of each drug alone. Antiproliferation was independent or nearly so (60% to 167% of prediction) for 14/18 combinations with one or both drugs at their lesser concentrations (bold values). Except in H1437, the combination S5E10 was more potent than either single agent, but weaker than predicted by independence. We speculate that EGFR and c-Met signaling pathways overlap in NSCLC, or E10 and S5 share off-target effects. Lack of pronounced synergism in drug-naive cells distinguishes de novo from acquired resistance to EGFR inhibition. We conclude that combined use of EGFR and c-Met inhibitors is rational even in front-line NSCLC therapy, if dose-limiting toxicities of the drugs differ. Relative proliferation of NSCLC lines incubated with erlotinib (E) and/or SU11274 (S). Cell Line S1 S5 E2 E10 S1E2 S5E2 S1E10 S5E10 A549 0.74 0.04 0.72 0.26 0.52 0.13 0.29 0.08 Calu3 0.88 0.23 0.16 0.15 0.36 0.22 0.18 0.10 H1299 0.81 0.18 0.66 0.24 0.63 0.20 0.25 0.14 H1437 0.97 0.10 0.90 0.37 0.58 0.20 0.46 0.12 H1975 0.65 0.10 0.65 0.23 0.61 0.04 0.23 0.06 H2122 0.64 0.07 0.66 0.26 0.44 0.06 0.13 0.04 Proliferation P = # cells drug/# cells DMSO. E2 = 2 uM erlotinib, etc. 1 SD = 4–11% of P. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration OSI Oncology

Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 μmol l−1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.

CD4 −CD8 − splenic T cells from lewis rats recovered from experimental autoimmune encephalomyelitis respond to encephalitogenic T cells that mediate this disorder

We previously demonstrated that encephalitogenic CD4 + T lymphocytes from the long-term cultured line S1, specific for myelin basic protein, induce a CD8 + T cell population in vivo that protects naive Lewis rats against experimental autoimmune encephalomyelitis caused by S1 cells. In order to determine the contribution of individual T cell population in the development of induced resistance, we have analyzed the in vitro proliferative capacity of phenotypically distinct T cell populations isolated from S1-immunized rats. We found that both CD8 + and CD8 −CD4 − T cells show striking proliferative responses when stimulated with S1 cells, whereas CD4 + T cells show only minimal responses. In addition, a significant proportion of the CD8 −CD4 − cells, after stimulated by S1 cells, became CD8 + and had a strong cytolytic activity toward S1 cells. These results suggest a contribution of double-negative splenic T cells in the regulatory circuit associated with autoimmune encephalomyelitis.

Evidence for Maternal Regulation of Progesterone Production at Midpregnancy in the Mouse

Mice with major differences in embryo survival and progesterone secretion were used to study the relative roles of maternal and embryonic genotype in the control of progesterone production at midpregnancy. Reciprocal transfer of embryos from strains with high (Line S1) or low (Line G) embryo survival resulted in progesterone concentrations consistent with maternal genotype in females carrying the same number of fetuses. This suggests that maternal rather than fetal gene expression determines the setpoint for progesterone production during the period of transition from maternal to fetal-placental control of ovarian steroidogenesis.

Full length L1 retroposons contain tRNA-like sequences near the 5′ termini-hypothesis on the replication mechanism of retroposons

Retrotransposons replicate via a complex mechanism which depends on, among other things, the presence of long terminal repeats (LTRs) and a tRNA binding site just 3' of the 5' LTR. The LINES 1 (L1) family of sequences, which similar to retrotransposons in many other properties, represents a new class of retroposon which does not possess LTRs. However, we show here that the repetitive 5' motif associated with murine L1 elements contains a tRNA-like sequence in a location analogous to the position of the retro-transposon tRNA binding site. Although the repetition of such a 5' motif has only been found associated with murine L1 elements, we have found an analogous tRNA-like sequence near the 5' ends of the L1 elements from each of the other analyzed species for which the L1 family has been characterized, that is rat (L1Rr), human (L1Hs), drosophila (I element) and trypanosome (INGI). The conservation of this tRNA-like sequence near the 5' terminus of L1 elements from such diverse species suggests that it plays a functional role in the life of the L1 class of retroposon.

Suppression of experimentally induced autoimmune encephalomyelitis by cytolytic T-T cell interactions.

Down-regulatory phenomena have been described in several experimental models of tissue-specific, T-cell-mediated autoimmunity. For example, resistance to active induction of experimental autoimmune encephalomyelitis (EAE) can be induced by pretreating animals with non-pathogenic inocula of autoantigen or effector cells. Moreover, animals that have recovered from one EAE episode are resistant to subsequent induction of EAE. In some models, resistance to EAE has been transferred with immune cells to naive recipients. These experiments, which were based on transfers of unseparated immune cell populations, are difficult to interpret. Immune suppression circuits are known to be complex and involve various distinct cellular subsets. To further complicate the issue, resistance to EAE can be transferred not only by suppressor cells, but also by encephalitogenic effector cells injected in 'subclinical' doses. We describe now the isolation of homogeneous T lymphocyte lines from the spleens of Lewis rats that had recovered from T-cell-mediated EAE (tEAE) caused by the MBP-specific T cell line S1. These spleen-derived T line cells express the CD8 phenotype and specifically respond to determinants on the inducing S1 line, but not to the autoantigen MBP. Furthermore, the anti-S1 cells selectively lyse the encephalitogenic S1 T line in vitro and efficiently neutralize their encephalitogenic capacity in vivo.

Male and female genotype mediate pheromonal regulation of the mouse estrous cycle.

Estrous cyclicity was studied to examine the possibility that strain differences in the regularity of the mouse estrous cycle are the result of different olfactory signals produced by the male. Females with regular estrous cycles (lines E and S1) were housed in the olfactory presence of males from a line with irregular cycles (line CN-) or in the presence of males of their own line (used as a control). Females with irregular cycles (line CN-) were housed in the presence of males from a line with regular cycles (line E) or were exposed to males of their own line. The regularity of the estrous cycle decreased in line E females (regular cycles) when exposed to line CN- males (irregular cycles). The decreased regularity of line E cyclicity resulted from an increased period of diestrus, i.e., lengthening of the cycle. In contrast, line S1 females (regular cycles) did not show any change in estrous cyclicity when exposed to line CN- males. The period of diestrus increased in line CN- females when they were exposed to line E males. These results provide evidence that 1) the genotype of the male can influence the regularity of the estrous cycle, and 2) the genotype of the female regulates her responsiveness to environmental factors (e.g., male odor).

Changes in the kinetics of follicular growth in response to selection for large litter size in mice.

The present study examined a randomly selected control line (C) and a large litter size-selected line of mice (S1) to determine changes in the kinetics of follicular growth that have occurred in response to selection for large litter size. For each follicle type (FT), the number of healthy and atretic follicles, length of components of granulosa cell cycle, follicular growth rate, and follicular flux were determined microscopically from serially sectioned ovaries of Lines C and S1 mice. Selection for litter size significantly increased the number of small and medium, and some large follicle-size classes. While selection for litter size did not change the overall incidence of atresia at proestrus, it did decrease the incidence of atresia in the large Type 7 follicles by 19%. Selection for litter size also increased ovarian weight at proestrus. Selection for litter size increased the rate of growth through FT 3a, 5a, and 5b, and reduced the time required for follicles to grow from primordial to Graafian follicles from 39.1 days in Line C to 33.4 days in Line S1. Selection for large litter size also increased the flux of follicles through follicle Types 3a to 5b by 72%, and through follicle Types 6 and 7 by 21%. Genetic variation was found in many aspects of the kinetics of follicular growth.

Experimental observation of the intercollisional interference effect on the S1 pure rotation line of the collision induced spectra of the H2-He mixture

The absorption coefficients of the collision induced spectrum of the interaction H2-He at 300 K between 200 and 700 cm-1 are presented. A lack of absorption (dip) due to the intercollisional interference effect is clearly seen at the frequency of the pure rotation line S1 of the free molecule of H2.

Genetic analysis of a strain of mice plateaued for litter size.

A strain of mice (S1) was successfully selected for large litter size for 31 generations, increasing the mean by 4.2 young per litter. After generation 31, there was no further progress and it was concluded that a selection plateau had been reached. Realized heritability decreased during the course of the experiment from 0.16+/-0.06 for the first 15 generations to 0.00+/-0.03 for generations 30 through 45.--In order to explore the nature of the selection plateau, the following groups were derived from line S1 at generation 34 or 35: Upward selection with inbreeding (SF), random (relaxed) selection (SO), and downward selection (SR). Selections were carried out for 10 to 11 generations. The means of SO and SF were similar to those of S1, ruling out any major effect of natural selection or overdominance. SR decreased, the mean averaging 2.3 young per litter below that of S1 during the last three generations. The fact that SR responded to selection indicates that genetic variance was still present in the plateaued population. The SF sublines were crossed when the inbreeding was 95% and a new line, SX, was formed. SX was maintained for three generations and the difference of +0.7 young per litter above the contemporary generations of S1 was significant. The results from this experiment suggest that the selection plateau in line S1 was caused by reduction of additive genetic variance to a very low level. Some nonadditive genetic variance remained, however, and was attributed to recessive alleles at low frequency. In agreement with results reported by Falconer (1971), inbreeding with selection followed by crossing of the inbred sublines proved to be effective in overcoming a selection plateau in litter size.