Human acute myeloid leukemia cells express Neurokinin-1 receptor, which is involved in the antileukemic effect of Neurokinin-1 receptor antagonists.

The substance P/neurokinin-1 receptor system has been implicated in tumor cell proliferation. Neurokinin-1 receptor has been identified in different solid tumors but not frequently in hematopoietic malignant cells. We investigated the presence of the Neurokinin-1 receptor in acute myeloid leukemia cell lines (KG-1 and HL-60), demonstrating that acute myeloid leukemia cell lines overexpress the truncated Neurokinin-1 receptor isoform compared with lymphocytes from healthy donors. Using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we demonstrated that substance P induced cell proliferation in both acute myeloid leukemia cell lines. We also observed that four different Neurokinin-1 receptor antagonists (L-733,060, L-732,138, CP 96-345 and aprepitant) elicited inhibition of acute myeloid leukemia cell growth lines in a concentration-dependent manner, while growth inhibition was only marginal in lymphocytes; the specific antitumor action of Neurokinin-1 receptor antagonists occurs via the Neurokinin-1 receptor, and leukemia cell death is due to apoptosis. Finally, administration of high doses of daily intraperitoneal fosaprepitant to NOD scid gamma mice previously xenografted with the HL60 cell line increased the median survival from 4 days (control group) to 7 days (treated group) (p = 0.059). Taken together, these findings suggest that Neurokinin-1 receptor antagonists suppress leukemic cell growth and may be considered to be potential antitumor drugs for the treatment of human acute myeloid leukemia.

Carotid body oxygen sensing.

The carotid body (CB) is a neural crest-derived organ whose major function is to sense changes in arterial oxygen tension to elicit hyperventilation in hypoxia. The CB is composed of clusters of neuron-like glomus, or type-I, cells enveloped by glia-like sustentacular, or type-II, cells. Responsiveness of CB to acute hypoxia relies on the inhibition of O(2)-sensitive K(+) channels in glomus cells, which leads to cell depolarisation, Ca(2+) entry and release of transmitters that activate afferent nerve fibres. Although this model of O(2) sensing is generally accepted, the molecular mechanisms underlying K(+) channel modulation by O(2) tension are unknown. Among the putative hypoxia-sensing mechanisms there are: the production of oxygen radicals, either in mitochondria or reduced nicotinamide adenine dinucleotide phosphate oxidases; metabolic mitochondrial inhibition and decrease of intracellular ATP; disruption of the prolylhydroxylase/hypoxia inducible factor pathway; or decrease of carbon monoxide production by haemoxygenase-2. In chronic hypoxia, the CB grows with increasing glomus cell number. The current authors have identified, in the CB, neural stem cells, which can differentiate into glomus cells. Cell fate experiments suggest that the CB progenitors are the glia-like sustentacular cells. The CB appears to be involved in the pathophysiology of several prevalent human diseases.

The NK1 receptor is involved in the antitumoural action of L-733,060 and in the mitogenic action of substance P on neuroblastoma and glioma cell lines.

We have carried out an in vitro study to investigate the ability of substance P to activate cell growth and the NK1 receptor antagonist L-733,060 to inhibit cell growth in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines. A coulter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. Nanomolar concentrations of substance P increased, and micromolar concentrations of L-733,060 inhibited the growth of both cell lines studied, with and without previous administration of substance P. In addition, we have demonstrated by immunoblot analysis that NK1 receptors are present in both cancer cell lines studied here. Thus, this study demonstrates that substance P acts as a mitogen in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines, and that the antitumoural action of L-733,060 on both human cell lines occurs through the NK1 receptor. This action suggests that the NK1 receptor is a new and promising target in the treatment of human neuroblastoma and glioma.

A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation-associated recombination.

We have identified two novel yeast genes, THO1 and THO2, that partially suppress the transcription defects of hpr1Delta mutants by overexpression. We show by in vivo transcriptional and recombinational analysis of tho2Delta cells that THO2 plays a role in RNA polymerase II (RNA pol II)-dependent transcription and is required for the stability of DNA repeats, as previously shown for HPR1. The tho2Delta mutation reduces the transcriptional efficiency of yeast DNA sequences down to 25% of the wild-type levels and abolishes transcription of the lacZ sequence. In addition, tho2Delta causes a strong increase in the frequency of recombination between direct repeats (>2000-fold above wild-type levels). Some DNA repeats cannot even be maintained in the cell. This hyper-recombination phenotype is dependent on transcription and is not observed in DNA repeats that are not transcribed. The higher the impairment of transcription caused by tho2Delta, the higher the frequency of recombination of a particular DNA region. The tho2Delta mutation also increases the frequency of plasmid loss. Our work not only identifies a novel yeast gene, THO2, with similar function to HPR1, but also provides new evidence for transcriptional blocks as a source of recombination. We propose that there is a set of proteins including Hpr1p and Tho2p, in the absence of which RNA pol II transcription is stalled or blocked, causing genetic instability.

The yeast HRS1 gene is involved in positive and negative regulation of transcription and shows genetic characteristics similar to SIN4 and GAL11.

We provide genetic evidence that HRS1/PGD1, a yeast gene previously identified as a suppressor of the hyper-recombination phenotype of hpr1, has positive and negative roles in transcriptional regulation. We have analyzed three differently regulated promoters, GAL1, PHO5 and HSP26, by beta-galactosidase assays of lacZ-fused promoters and by Northern analysis of the endogenous genes. Transcription of these promoters was derepressed in hrs1delta mutants under conditions in which it is normally repressed in wild type. Under induced conditions it was either strongly reduced or significantly enhanced depending on the promoter system analyzed. Constitutive transcription was not affected, as determined in ADH1 and TEF2. In addition, Hrs1p was required for mating-factor expression, telomere-linked DNA silencing and DNA supercoiling of plasmids. Furthermore, hrs1delta suppressed Ty-insertion mutations and conferred a Gal- phenotype. Many of these phenotypes also result from mutations in GAL11, SIN4 or RGR1, which encode proteins of the RNA polII mediator. We also show that gal11delta and sin4delta partially suppress the hyper-rec phenotype of hpr1 mutants, although to a lesser extent than hrs1delta. Our results provide new evidence for the connection between hpr1delta-induced deletions and transcription. We discuss the possibility that Hrs1p might be a component of the RNA polII transcription machinery.

Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation.

The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes. Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination. Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription. We show that the induction of deletions between repeats in hpr1delta cells of Saccharomyces cerevisiae is linked to transcription elongation. Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1delta are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats. Transcription becomes HPR1 dependent when elongating through such regions. Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats. In contrast to previously reported cases of transcription-induced recombination, there was no correlation between high levels of transcripts and high levels of recombination. Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation.

Mutations in the yeast SRB2 general transcription factor suppress hpr1-induced recombination and show defects in DNA repair.

We have obtained genetic and molecular evidence that the hrs2-1 mutation, isolated as a suppressor of the hyperrecombination phenotype of hpr1 delta, is in the SRB2 gene, which encodes a component of the RNA polII holoenzyme. A newly constructed srb2 delta allele restores the wild-type levels of deletions in hpr1 delta cells, indicating that the lack of a functional SRB2 transcription factor suppresses recombination between direct repeats. These results suggest a direct connection between transcription and recombination between DNA repeats. On the other hand, the hrs2-1 mutation (renamed srb2-101), in which Gly150 has been changed to Asp, makes cells sensitive to long MMS treatments, a phenotype observed for the srb2 delta null allele only in a hpr1 delta background. This indicates that mutations in the basal transcription factor SRB2 impair DNA repair of MMS-induced damage, which adds a new connection between transcription and DNA repair. We discuss the possibility that hpr1-induced deletions occurred as a consequence of a SRB2-dependent stalled or blocked transcription complex.