Nikola Valkov

Histone deacetylase interacts directly with DNA topoisomerase II

Histone deacetylases (HDACs) modify nucleosomal histones, have a key role in the regulation of gene transcription, and may be involved in cell-cycle regulation, differentiation and human cancer. Purified recombinant human HDAC1 protein was used to screen a cDNA expression library, and one of the clones identified encoded DNA topoisomerase II (Topo II), an enzyme known to have a role in transcriptional regulation and chromatin organization. Coimmunoprecipitation experiments indicate that HDAC1 and HDAC2 are associated with Topo II in vivo under normal physiological conditions. Complexes containing Topo II possess HDAC activities, and complexes containing HDAC1 or HDAC2 possess Topo II activities. HDAC and Topo II modify each others activity in vitro and in vivo. Our results indicate the existence of a functionally coupled complex between these two enzymes and offer insights into the potential mechanisms of action of both enzymes.

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose.

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.

Effects of wild‐type p53 expression on the quantity and activity of topoisomerase IIα and β in various human cancer cell lines

The p53 null HL‐60 cell line was transfected with plasmids coding for either the wild‐type p53 or mutant p53 gene. The stable expression of wild‐type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR‐529. HL‐60 cells expressing wild‐type p53 demonstrated 8‐ to 10‐fold more VP‐16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild‐type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild‐type p53 in the K562 cell line resulted in a 3‐fold increase in sensitivity to VP‐16. The quantity of topoisomerase IIα was not altered by the transfection as determined by immunoblotting, while the amount of the β isoform was increased 2.5‐fold in HL‐60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIα was substantially diminished in both stable and inducible wild‐type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP‐16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIα. No changes in topoisomerase IIβ immunostaining were observed. These results suggest that an epitope for topoisomerase IIα is concealed in cells expressing wild‐type p53 and that a complex between topoisomerase IIα and p53 may be disrupted by the addition of antitumor drugs. J. Cell. Biochem. 75:245–257, 1999.

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild‐type and drug‐resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug‐resistant SMR16 and parental cells in the presence and absence of VP‐16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole‐mount spreading of residual structures permits, by means of isoform‐specific antibody and colloidal‐gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild‐type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole‐mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997.

Enhanced cell accumulation and collagen processing by keratocytes cultured under agarose and in media containing IGF-I, TGF-β or PDGF.

We previously showed an agarose overlay on keratocytes cultured in media containing pharmacological levels of insulin enhanced collagen processing and collagen fibril formation. In this study, we compared collagen processing by keratocytes cultured in media containing physiological levels of IGF-I, TGF-β, FGF-2, and PDGF in standard and in agarose overlay cultures. Pepsin digestion/SDS PAGE was used to determine the levels of procollagen secreted into the media and the collagen content of the ECM associated with the cell layer. Distribution of collagen type I and fibronectin in the ECM of the agarose cultures was determined by immunoflorescence. Collagen fibril and keratocyte morphology was evaluated by electron microscopy. The agarose overlay significantly enhanced the cell number in the IGF-I, TGF-β and PDGF treated cultures by 2-3 fold. The overlay also significantly enhanced the processing of procollagen to collagen fibrils from 29% in standard cultures to 63-68% in agarose cultures for the IGF-I and PDGF cultures, and from 66% in standard culture to 85% in agarose culture for the TGF-β cultures. Cell accumulation and collagen processing was not enhanced by agarose overlay of the FGF-2 treated cultures. Collagen type I and fibronectin were more uniformly distributed and the collagen fibrils smaller in the ECM of the TGF-β treated cultures. Keratocytes in the FGF-2 treated cultures were in close cell contact with few collagen fibrils while IGF-I, TGF-β, and PDGF cultures had an extensive ECM with abundant collagen fibrils. The results of this study indicate that the agarose overlay enhances collagen fibril assembly and cell accumulation by keratocytes when both collagen synthesis and cell proliferation are stimulated.

Sampling strategies for tissue microarrays to evaluate biomarkers in ovarian cancer.

Introduction: Tissue microarrays (TMA) enable rapid analysis of biomarkers in large-scale studies involving archival tumor specimens, however, their utility in heterogeneous tumors such as ovarian cancer is limited. Methods: In this study, immunohistochemical analysis was done on TMAs comprised of epithelial ovarian cancer (EOC) to estimate the prevalence of loss of expression of three mismatch repair proteins. TMAs were initially created using cores sampled from the center of donor tissue blocks from 59 EOC cases. Full sections were subsequently created and levels of expression were compared between tissues sampled from the central portion versus the periphery. Follow-up analyses were done by obtaining cores from the periphery of up to five additional donor blocks per case. A linear mixed model for each protein was used to investigate differences between results from the initial and follow-up blocks. Results: In the original TMAs created using centrally sampled cores, loss of mismatch repair expression was noted in 17 (29%) of the 59 cases. By comparison, analyses from peripherally sampled cores revealed loss of expression in only 6 of these 17 cases. For each protein, significant differences (P < 0.05) were detected between results from the initial donor block and the majority of the follow-up blocks. Conclusions: Our investigations, based on EOC, suggest that sampling variability in protein expression may result when TMAs are used. Thus, at least for EOC, it is important to preferentially sample from the periphery of tumor blocks where exposure to tissue fixatives is optimal. (Cancer Epidemiol Biomarkers Prev 2009;18(1):28–34)