The secretory KCa1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence.

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na(+) absorption and K(+) secretion. The electrogenic Na(+) absorption is mediated by epithelial Na(+) channel (ENaC) in surface cells. Previously, we identified the large conductance Ca(2+)-activated K(+) channel, K(Ca)1.1 or big potassium (BK) channel, as the only relevant K(+) secretory pathway in mouse distal colon. The exact localisation of K(Ca)1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K(Ca)1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na(+)/K(+)/2Cl(-) (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K(Ca)1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K(Ca)1.1 α-subunit were, however, under these circumstances substantially augmented (K(Ca)1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na(+) absorption and BK channel-mediated K(+) secretion are two independent processes. These findings show that K(Ca)1.1-mediated K(+) secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.

A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity.

Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.

Nutrient mixture including vitamin C, L-lysine, L-proline, and epigallocatechin is ineffective against tumor growth and metastasis in a syngeneic neuroblastoma model.

BACKGROUND: The replacement of established evidence-based cancer therapy protocols (mainstream therapy) by unevaluated complementary and alternative medicine (CAM) is a challenge in pediatric oncology. We tested the hypothesis that oral application of L-lysine and ascorbic acid (Lysin C Drink) in combination with epigallocatechin-gallate (EGCG) and amino-acids (Epican forte) is effective in a preclinical model of neuroblastoma. METHODS: Primary tumors and spontaneous metastases were induced in A/J mice by injection of NXS2 neuroblastoma cells. Mice were treated by daily oral gavage with L-lysine and ascorbic acid (Lysin C Drink) (equivalent to 150 mg ascorbic acid/day/mouse) (treatment A) or with EGCG plus ascorbic- and amino-acids (Epican forte) (9.2 mg/mouse) (treatment B). Treatment A was started in the prophylactic setting (7 days before tumor cell injection) as well as in the therapeutic setting (1 day after tumor cell inoculation). Finally, treatment B was evaluated alone and in combination with treatment A in the therapeutic setting. The effect on primary tumor growth and the development of spontaneous liver metastases was evaluated. RESULTS: L-lysine and ascorbic acid (Lysin C Drink) and EGCG plus ascorbic- and amino-acids (Epican forte) are ineffective in reduction of primary tumor growth and prevention of spontaneous liver metastases in this model. CONCLUSIONS: Neither a formal clinical development nor the use of these substances can be recommended for neuroblastoma.

Characterization of GD2 peptide mimotope DNA vaccines effective against spontaneous neuroblastoma metastases.

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma.

MCM2 promotes symmetric inheritance of modified histones during DNA replication.

During genome replication, parental histones are recycled to newly replicated DNA with their posttranslational modifications (PTMs). Whether sister chromatids inherit modified histones evenly remains unknown. We measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading-strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones are inherited to sister chromatids and identifies a mechanism by which the replication machinery ensures symmetric cell division.

A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.