Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2

Proliferation and cell death are regarded as key targets for the optimization of animal cell culture processes and for the maximization of product yield. Although chemical and physical factors are vitally important, of primary interest is the utilization of genetic engineering to regulate cellular processes. CHO cells were first genetically modified to enhance proliferation rate in both suspension and monolayer cultures. Under the constitutive control of c-myc overexpression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Although the c-myc transfected cell line exhibited apoptosis at much lower rates than is widely reported and associated with the overexpression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, to reduce cell death. Overexpression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line. In conclusion, it was shown that Bcl-2 protein overexpression specifically abrogates c-Myc-induced apoptosis without affecting the c-Myc mitogenic function.     

A switch in pdgfrb+ cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord

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Comparative immunostaining of T-associated plasma cells and other lymph-node cells in paraffin sections

The immunoperoxidase technique after trypsinization was used on paraffin sections of 24 lymph nodes with reactive lymphadenitis and abundant nests of T-associated plasma cells. These cells were negative for all the markers investigated, which were: intracytoplasmic immunoglobulins (CIg), a1-antichymotrypsin(a1-ACT), a1-antitrypsin(a1-AT), lysozyme(Lz) and fibronectin. Other categories of cells were positive or negative depending on their type. The best markers for polymorphs proved to be a1-AT and Lz, and for monocytes and histiocytes a1-ACT and Lz. Sinus histiocytes in particular were much more constantly and strongly positive for a1-ACT than for Lz. Endothelial cells appeared almost always positive for a1-ACT and were also occasionally positive for a1-AT. Fibronectin was consistently positive in mast cells and sometimes positive in other cells, especially those of monohistiocytic origin. Our present findings are against a B-cell or monohistiocytic origin for T-associated plasma cells.     

TET2 is a component of the estrogen receptor complex and controls 5mC to 5hmC conversion at estrogen receptor cis-regulatory regions

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Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are active at resting membrane potential and thus are likely to contribute to neuronal excitability. Four HCN channel subunits (HCN1–4) have previously been cloned. The aim of the current study was to investigate the immunoreactivity of HCN4 channel protein in rat trigeminal (TG) and dorsal root ganglion (DRG) sensory neurons. HCN4 was present in 9% of TG neurons and 4.7% of DRG neurons, it was distributed in a discrete population of small-diameter neurons in the TG but was located in cells of all sizes in the DRG. Approximately two thirds of HCN4-containing neurons in each ganglia were labelled with antisera raised against the 200-kDa neurofilament (NF200). The remaining HCN4-containing neurons were NF200-negative, were not labelled with antisera raised against calcitonin-gene related peptide (CGRP), and did not bind the isolectin B4 (IB4). HCN4-containing neurons made up more than half of the population of small-diameter primary afferent neurons that did not contain either NF200 or CGRP or bind IB4 in both TG and DRG. This population was not insignificant, comprising 5% of TG neurons and 2% of DRG neurons.     

Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2‐DE and MS led to the generation of their 2‐DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2‐DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.