Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment.

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Neurospheres derived from human embryoid bodies treated with retinoic Acid show an increase in nestin and ngn2 expression that correlates with the proportion of tyrosine hydroxylase-positive cells.

In the central nervous system (CNS), generation of phenotypic diversity within the neuronal lineage is precisely regulated in a spatial and temporal fashion. Neural basic helix-loop-helix (bHLH) transcription factors are cell intrinsic factors that control commitment to neuronal lineage and play an important role in neuronal cell type specification. The ability to differentiate human embryonic stem (hES) cells into neurons provides a good model system to address human neuronal specification. Previous studies have shown neurogenin-2 (Ngn2) to be involved in the development of mesencephalic dopaminergic neurons. Toward the goal of correlating neuronal phenotype with early gene expression pattern, we have characterized the expression of Ngn2 during hES cell differentiation. Our results show that treatment of embryoid bodies (EBs) with retinoic acid (RA) leads to the greatest proportion of tyrosine hydroxylase (TH)-positive cells followed by vasoactive intestinal peptide (VIP)-treated EBs as compared to untreated EBs. This increase in the proportion of TH-positive neurons was correlated with the unique morphology of RA-treated aggregates and the spatial delocalization of the expression of Ngn2 within the EB. Neurospheres derived from RA-treated EBs contained many nestin-positive cells within regions that expressed Ngn2. We show that the extent of nestin-positive cells that arise from the region of Ngn2 expression is correlated with the appearance of TH-positive neurons. Our results show for the first time the expression of Ngn2 during the differentiation of hES cells.