Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cellcycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57 KIP2 (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G 1 as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated ␤-galactosidase marker within all astrocytoma cell lines such that ϳ75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells (ϳ15%) that were nonviable, contained discrete nuclear fragments on The most common brain tumor is the astrocytoma accounting for ϳ65% of all primary brain tumors. The malignant astrocytoma has a very poor prognosis primarily because of its highly proliferative and invasive nature. As with other neoplasms with increased proliferative potential, malignant astrocytomas demonstrate dysregulation of various components of the cell cycle machinery. Altered expression of positive growth regulators such as growth factors, cyclins, and cyclin-dependent kinases (CDKs), or the loss of negative regulators, including cyclin-dependent kinase inhibitors (CKIs) and the retinoblastoma protein (pRB) have all been demonstrated in malignant astrocytomas. 1,2 The CDKs phosphorylate pRB to release cells from cell-cycle arrest. In contrast with CDKs, the CKIs inhibit cyclin-CDK complexes and transduce internal or external growth suppressive signals. Accordingly, all CKIs may be construed as candidate tumor suppressor genes.The CKIs are divided into two families, the INK4 and the CIP/KIP, which are defined on the basis of their structural homology and mechanism of action. The CIP/KIP family includes three structurally related members, p21 CIP1/WAF1 , 3,4 p27 KIP1 , 5,6 and a recently isolated and cloned third member, p57 KIP2 (p57). 7-10 These three CKIs share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. Mouse p57 consists of four structurally distinct domains, a CDK inhibitory domain, a proline-rich domain, an a...

No abstract

A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

Intermediate filaments (IFs) are highly diverse intracytoplasmic proteins within the cytoskeleton which exhibit cell type specificity of expression. A growing body of evidence suggests that IFs may be involved as collaborators in complex cellular processes controlling astrocytoma cell morphology, adhesion and proliferation. As the co-expression of different IF subtypes has been linked to enhanced motility and invasion in a number of different cancer subtypes, we undertook the present study to examine the expression of vimentin and nestin in a panel of human astrocytoma cell lines whose tumorigenicity, invasiveness and cytoskeletal protein profiles are well known. Astrocytoma cells were examined for IF protein expression by immunofluorescence confocal and immunoelectron microscopy. The motility of all cell lines was determined by computerized time-lapse videomicroscopy. Invasive potential of astrocytoma cells was determined using Matrigel as a barrier to astrocytoma cell invasion in vitro. Vimentin was expressed by all astrocytoma cell lines. On the other hand, nestin was variably expressed among the different cell lines. The most motile and invasive astrocytoma cell line in our study was antisense GFAP-transfected U251 (asU251) astrocytoma cells which showed marked up-regulation of nestin expression compared to the U251 parental cell line and controls. The U87 astrocytoma cell line also demonstrated high nestin expression levels and was associated with an increased basal motility rate and a high degree of invasiveness through Matrigel. U343 astrocytoma cells did not express nestin, but had high levels of GFAP. It had the lowest motility rate and invasiveness of all the astrocytoma cell lines examined. Taken together, these data suggest that for the astrocytoma cell lines examined in this study, nestin and vimentin co-expression may serve as a marker for an astrocytoma cell type with enhanced motility and invasive potential. Further studies are required to determine the mechanism by which dual-IF protein expression alters other cytoskeletal or cell surface receptor protein components important in the process of astrocytoma invasion.