Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/−/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611

We have previously generated a mouse model (Ptch1+/−/Tis21KO), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/−/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/− or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/−/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/−/Tis21KO MBs, relative to Ptch1+/−/Tis21WT MBs. In Ptch1+/−/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/−/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/−/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.

The Large pBS32/pLS32 Plasmid of Ancestral Bacillus subtilis

ABSTRACT The ancestral strain of Bacillus subtilis NCIB3610 (3610) bears a large, low-copy-number plasmid, called pBS32, that was lost during the domestication of laboratory strain derivatives. Selection against pBS32 may have been because it encodes a potent inhibitor of natural genetic competence (ComI), as laboratory strains were selected for high-frequency transformation. Previous studies have shown that pBS32 and its sibling, pLS32 in Bacillus subtilis subsp. natto, encode a replication initiation protein (RepN), a plasmid partitioning system (AlfAB), a biofilm inhibitor (RapP), and an alternative sigma factor (SigN) that can induce plasmid-mediated cell death in response to DNA damage. Here, we review the literature on pBS32/pLS32, the genes found on it, and their associated phenotypes.

Genetic Transformation of Plumbago zeylanica with Agrobacterium rhizogenes Strain LBA 9402 and Characterization of Transformed Root Lines

High frequency transformation (73.80 ± 2.24%) has been obtained in Plumbago zeylanica using nodes and internodes of axenic whole plants infected with Agrobacterium rhizogenes strain LBA 9402. The root lines could be distinguished morphologically into two types : Root lines of morphotypes I and II. While morphotype I showed profuse branching with short (< 1 cm), highly dense hairy laterals, the roots of morphotype II roots were characterized also by profuse branching with long hairy laterals (> 3 ? 4 cm). Only four of the ten root lines showed integration of four rol genes (rolA, rolB, rolC and rolD) of TL?DNA. None of the root lines showed presence of any of the five genes of TR?DNA. It is noteworthy that the root morphotypes (I and II) showed a clear distinction in the nature of integration and expression of rol genes. The transformed root lines varied significantly (p ? 0.05) with respect to DW (GI DW basis, 2.19 ± 0.24 ? 5.31 ± 0.6) after 4 weeks of culture on solid modified MS; and plumbagin contents in root lines (4.81 ± 0.16 ? 6.69 ± 0.34 mg/g DW) were higher than that reported earlier. Transformed root lines of P. zeylanica maintained in vitro on phytohormone devoid medium for over 2 years can be used for scale up studies for the production of plumbagin in bioreactors.Plant Tissue Cult. & Biotech. 25(1): 21-35, 2015 (June)

Optimization of Regeneration Protocol of Rice from Embryo Derived Callus

An efficient and reproducible protocol for in vitro regeneration is required to achieve high frequency transformation from transformed calli. We report here high frequency plant regeneration from mature embryonic calli of two BINA rice cultivars Binasail and Binadhan-4. Embryonic calllus initiated on MS basal medium supplemented with 2 mg/l 2, 4-D. Several media with different combinations of growth regulators were tried. Maximum shoot regeneration frequency (63.33%) was observed in Binadhan-4 on MS medium supplemented with 5 mg/l Kinetin + 0.5 mg/l NAA. Maximum root regeneration frequency (70.00%) was observed in Binadhan-4 on MS medium supplemented with 6 mg/l Kinetin + 0.5 mg/l NAA. Well developed plantlets were hardened and transferred to the glasshouse.DOI: http://dx.doi.org/10.3329/pa.v18i2.17461 Progress. Agric. 18(2): 25 - 33, 2007