ABSTRACT
Recent evidence supports the hypothesis that cancer stem cells are responsible for tumour initiation and formation. Using flow cytometry, we isolated a population of CD44+CD24(-) prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44+CD24(-) cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44+CD24(-) cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44+CD24(-) prostate stem-like cells as nonadherent spheres in serum-replacement media without substantially shifting gene expression. Addition of serum results in adherence to plastic and shifts gene expression patterns to resemble the differentiated parental cells. Thus, we propose that CD44+CD24(-) prostate cells are stem-like cells responsible for tumour initiation and we provide a genomic definition of these cells and the differentiated cells they give rise to. Furthermore, gene expression patterns of CD44+CD24(-) cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44+CD24(-) LNCaP prostate cells offer an attractive model system to both explore the biology important to the maintenance and differentiation of prostate cancer stem cells as well as to develop the therapeutics, as the gene expression pattern in these cells is consistent with poor survival in prostate cancer patients.
Subject(s)
Biomarkers, Tumor/metabolism , CD24 Antigen/metabolism , Hyaluronan Receptors/metabolism , Models, Biological , Neoplastic Stem Cells/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Animals , Biomarkers, Tumor/genetics , Flow Cytometry , Gene Expression Profiling , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Phenotype , Prognosis , Prostate/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , Tumor Stem Cell AssayABSTRACT
The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). Both antibodies recognized an antigen with the expected molecular weight of 93-95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GN(N) was markedly distinct from that labeled by anti-GN(C), a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48-50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis.
Subject(s)
Brain Chemistry/physiology , Brain/growth & development , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Calpain , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Humans , Hydrolysis , Immunoblotting , Immunohistochemistry , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Molecular Sequence Data , RNA, Messenger/biosynthesis , Rats , Telencephalon/metabolismABSTRACT
Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.
