Structural manifestation of the delocalization error of density functional approximations: C(4N+2) rings and C(20) bowl, cage, and ring isomers.

The ground state structure of C(4N+2) rings is believed to exhibit a geometric transition from angle alternation (N < or = 2) to bond alternation (N > 2). All previous density functional theory (DFT) studies on these molecules have failed to reproduce this behavior by predicting either that the transition occurs at too large a ring size, or that the transition leads to a higher symmetry cumulene. Employing the recently proposed perspective of delocalization error within DFT we rationalize this failure of common density functional approximations (DFAs) and present calculations with the rCAM-B3LYP exchange-correlation functional that show an angle-to-bond-alternation transition between C(10) and C(14). The behavior exemplified here manifests itself more generally as the well known tendency of DFAs to bias toward delocalized electron distributions as favored by Huckel aromaticity, of which the C(4N+2) rings provide a quintessential example. Additional examples are the relative energies of the C(20) bowl, cage, and ring isomers; we show that the results from functionals with minimal delocalization error are in good agreement with CCSD(T) results, in contrast to other commonly used DFAs. An unbiased DFT treatment of electron delocalization is a key for reliable prediction of relative stability and hence the structures of complex molecules where many structure stabilization mechanisms exist.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

Elevated C-peptide and insulin predict increased risk of colorectal adenomas in normal mucosa.

BACKGROUND: Lower concentrations of the insulin-like growth factor binding protein-1 (IGFBP-1) and elevated concentrations of insulin or C-peptide have been associated with an increase in colorectal cancer risk (CRC). However few studies have evaluated IGFBP-1 and C-peptide in relation to adenomatous polyps, the only known precursor for CRC. METHODS: Between November 2001 and December 2002, we examined associations between circulating concentrations of insulin, C-peptide, IGFBP-1 and apoptosis among 190 individuals with one or more adenomatous polyps and 488 with no adenomatous polyps using logistic regression models. RESULTS: Individuals with the highest concentrations of C-peptide were more likely to have adenomas (OR = 2.2, 95% CI 1.4-4.0) than those with the lowest concentrations; associations that appeared to be stronger in men (OR = 4.4, 95% CI 1.7-10.9) than women. Individuals with high insulin concentrations also had a higher risk of adenomas (OR = 3.5, 95% CI 1.7-7.4), whereas higher levels of IGFBP-1 were associated with a reduced risk of adenomas in men only (OR = 0.3, 95% CI 0.1-0.7). Overweight and obese individuals with higher C-peptide levels (>1(st) Q) were at increased risk for lower apoptosis index (OR = 2.5, 95% CI 0.9-7.1), an association that remained strong in overweight and obese men (OR = 6.3, 95% CI 1.0-36.7). Higher levels of IGFBP-1 in overweight and obese individuals were associated with a reduced risk of low apoptosis (OR = 0.3, 95% CI 0.1-1.0). CONCLUSIONS: Associations between these peptides and the apoptosis index in overweight and obese individuals, suggest that the mechanism by which C-peptide could induce adenomas may include its anti-apoptotic properties. This study suggests that hyperinsulinemia and IGF hormones predict adenoma risk, and that outcomes associated with colorectal carcinogenesis maybe modified by gender.

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.