Discovery of Power-Law Growth in the Self-Renewal of Heterogeneous Glioma Stem Cell Populations

Background

Accumulating evidence indicates that cancer stem cells (CSCs) drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS) forming model, to generate a population in which glioma stem cells (GSCs) become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability.

Methodology/Principal Findings

To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres). Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone). Log-log plots of distributions of clone sizes yielded a good fit (r>0.90) to a straight line (log(% total clones) = k*log(#cells/clone)) indicating that the system follows a power-law (y = x^k) with a specific degree exponent (k = −1.42). Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = −1.01 to −1.17). Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM), suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population.

Conclusions/Significance

Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous glioma stem cell populations. That the data always fit a power-law suggests that: (i) clone sizes follow continuous, non-random, and scale-free hierarchy; (ii) precise biologic rules that reflect self-organizing emergent behaviors govern the generation of neurospheres. That the power-law behavior and the original GS heterogeneity are maintained over multiple passages indicates that these rules are invariant. These self-organizing mechanisms very likely underlie tumor heterogeneity during tumor growth. Discovery of this power-law behavior provides a mechanism that could be targeted in the development of new, more effective, anti-cancer agents.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

Increased level of apolipoprotein B mRNA in the liver of ventromedial hypothalamus lesioned obese rats

The mRNA level of apolipoprotein B (apoB), which is a principal protein component of nascent very low density lipoprotein (VLDL), was determined in parallel with the measurement of acetyl-coenzyme A (Ac-CoA) carboxylase activity in the liver of ventromedial hypothalamus (VMH) lesioned obese rats. Eight weeks after the electrolysis of the bilateral VMH, the level of apoB mRNA in the VMH-lesioned rats was about 1.5-fold higher than that in the sham-operated rats, indicating increased apoB synthesis in the liver of the VMH-lesioned obese rats. The activity of Ac-CoA carboxylase, which is a rate-limiting enzyme for the fatty acid biosynthesis, was about 1.8-fold higher in the VMH-lesioned rats. These observations indicated that VLDL synthesis is increased in the liver of VMH-lesioned obese rats.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia

A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resulting gene silencing in human cancers. In addition, several gene promoters have also been shown to become hypermethylated in non-neoplastic cells during aging. To assess the physiological consequence and clinical significance of gene promoter methylation in gastric epithelia, our laboratory has studied the methylation status of tumor suppressor and tumor-related genes, including APC, DAP-kinase, DCC, E-cadherin, GSTP1, hMLH1, p16, PTEN, RASSF1A, RUNX3 and TSLC1, in neoplastic and non-neoplastic gastric epithelia. The tumor suppressor and tumor-related genes, except APC, were generally unmethylated in non-neoplastic gastric epithelia obtained from younger individuals. The frequencies of methylation increased with age to varying degrees in various genes, although GSTP1 and PTEN methylation was completely absent in both neoplastic and non-neoplastic gastric epithelia. The methylation frequencies in each gene were found to be comparable in neoplastic and non-neoplastic gastric epithelia, except the methylation of RUNX3 and TSLC1, which was mostly cancer-specific (P<0.01). When methylation frequencies were compared between non-neoplastic gastric epithelia from cancer-bearing and non-cancer-bearing stomachs, hMLH1 and p16 methylation was more frequent in those from cancer-bearing stomachs (P<0.01). Promoter methylation in tumor suppressor and tumor-related genes initially occurs in non-neoplastic gastric epithelia, increases with age, and ultimately silences gene function to constitute a field-defect that may predispose tissues to gastric cancer evolution. In clinical applications RUNX3 and TSLC1 methylation may be utilized as molecular diagnostic markers, and hMLH1 and p16 methylation as predictors of malignancy in the stomach.     

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Gene expression profile analysis of rheumatoid synovial fibroblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium

To elucidate the aberrant growth properties of rheumatoid synoviocytes, we have examined the gene expression profile of rheumatoid synovial fibroblasts (RSFs) and compared with that of normal synovial fibroblasts (NSF). Gene expression profile analysis was conducted with synoviocyte cultures obtained from five rheumatoid arthritis (RA) patients and five control cases using a commercial cDNA array containing the defined 588 cancer-related genes. The results were confirmed by real-time RT-PCR. Gene expression levels for the platelet-derived growth factor receptor alpha (PDGFRalpha), plasminogen activator inhibitor-1 (PAI-1), and stromal cell derived factor 1A (SDF1A) are constitutively augmented in RSF compared with NSF. The mRNA levels of PDGFRalpha, PAI-1, and SDF1A in RSF over NSF were 4.6-, 14-, and 2.8-fold, respectively, by real-time RT-PCR. In fact, we found that RSFs showed greater sensitivity to the cell proliferative effect of PDGF. T his aberrant gene expression profile suggests that RSF may have retained the premature phenotype of primordial synoviocytes.