Biomedical subjects

Nikolai N Khodarev

Publications and source records attributed to Nikolai N Khodarev.

8 recordsLinked to original sources

PubMed2005

Progression of Barrett's metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation.

We did expressional profiling on 24 paired samples of normal esophageal epithelium, Barrett's metaplasia, and esophageal adenocarcinomas. Matching tissue samples representing the three different histologic types were obtained from each patient undergoing esophagectomy for adenocarcinoma. Our analysis compared the molecular changes accompanying the transformation of normal squamous epithelium with Barrett's esophagus and adenocarcinoma in individual patients rather than in a random cohort. We tested the hypothesis that expressional profiling may reveal gene sets that can be used as molecular markers of progression from normal esophageal epithelium to Barrett's esophagus and adenocarcinoma. Expressional profiling was done using U133A GeneChip (Affymetrix), which represent approximately two thirds of the human genome. The final selection of 214 genes permitted the discrimination of differential gene expression of normal esophageal squamous epithelium, Barrett's esophagus, and adenocarcinoma using two-dimensional hierarchical clustering of selected genes. These data indicate that transformation of Barrett's esophagus to adenocarcinoma is associated with suppression of the genes involved in epidermal differentiation, including genes in 1q21 loci and corresponding to the epidermal differentiation complex. Correlation analysis of genes concordantly expressed in Barrett's esophagus and adenocarcinoma revealed 21 genes that represent potential genetic markers of disease progression and pharmacologic targets for treatment intervention. PCR analysis of genes selected based on DNA array experiments revealed that estimation of the ratios of GATA6 to SPRR3 allows discrimination among normal esophageal epithelium, Barrett's dysplasia, and adenocarcinoma.

Adenocarcinoma↗

PubMed2004

Interaction of amifostine and ionizing radiation on transcriptional patterns of apoptotic genes expressed in human microvascular endothelial cells (HMEC).

PURPOSE: Amifostine is a prodrug that requires dephosphorylation by alkaline phosphatase to become activated. This process occurs rapidly within the bloodstream after its i.v. administration to patients undergoing cancer treatment with selected radiation and chemotherapies. Vascular endothelial cells will, therefore, represent a normal cell system that is among the first to experience the radioprotective effects of this agent. Amifostine's active free thiol WR-1065 was investigated to determine its effect on radiation-induced changes in transcriptional patterns and subsequent apoptosis in human microvascular endothelial cells (HMEC) growing in vitro. METHODS AND MATERIALS: Human microvascular endothelial cells were grown to confluency and then exposed to WR-1065 at a concentration of 4 mM for 30 min, radiation doses that ranged from 0 to 6 Gy, and WR-1065 at a concentration of 4 mM for 30 min before exposure to ionizing radiation. Cell survival was assessed by clonogenic assay, cell cycle phase was analyzed by flow cytometry, apoptosis was also assessed by flow cytometry in which Anexin V staining and sub-G1 fraction analysis were applied, and gene expression was analyzed by the Clontech Atlas Human cDNA array to identify synergistic and antagonistic effects as a function of amifostine and radiation exposure conditions with a focus on apoptotic-related factors. RESULTS: Exposure of HMEC to 4 mM WR-1065 30 min before irradiation resulted in a protection enhancement factor of 2.0; that is, D(O-IRR) of 1.25 Gy and D(O-IRR+WR) of 2.56 Gy. Expression profiling revealed 29 genes that were synergistically activated by the combined action of WR-1065 and ionizing radiation, and an additional 12 genes were synergistically or additively suppressed. In particular, a subset of apoptosis-related genes that included caspases 2, 4, and 9 and different members of the bcl family, along with apoptosis-related receptors, were identified as being significantly affected by the combined treatment of WR-1065 and radiation exposure. In addition, a number of cell cycle-related genes that express cyclins A, G1, G2, and D3 and DNA damage/check point proteins ATM, DNA-PK and RAD23B were also found to be significantly affected. Functional assays of apoptosis were also performed that demonstrated the ability of WR-1065 to protect against radiation-induced apoptosis. CONCLUSIONS: WR-1065, the active thiol form of amifostine, is an effective radioprotector of HMEC as determined by use of clonogenic and apoptotic assays for cell survival. Expression profiling successfully defined the transcriptional response of HMEC to both WR-1065 and ionizing radiation exposure, either alone or in combination, and demonstrated both synergistic and antagonistic effects on the expression of different cellular genes, along with corresponding functional responses. The radioprotective effects of amifostine are not limited to its well-characterized physiochemical properties, which include free-radical scavenging, auto-oxidation leading to intracellular hypoxia, and chemical repair by hydrogen atom donation, but include its ability to modulate the complex transcriptional regulation of genes that are involved in apoptosis, cell cycle, and DNA repair.

Amifostine↗

PubMed2004

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells.

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.

Apoptosis↗

PubMed2004

Endothelial cells co-cultured with wild-type and dominant/negative p53-transfected glioblastoma cells exhibit differential sensitivity to radiation-induced apoptosis.

We performed expressional profiling of isogenic glioblastoma cell lines U87-Lux8 and U87-175.4. These cell lines differ in that U87-Lux8 expresses wild-type p53 and U87-175.4 expresses a dominant-negative p53 (175(His) mutation). DNA array analysis and real-time PCR measurements demonstrated that basal expression and response to irradiation were different in these isogenic glioblastoma cell lines. These differences included genes involved in growth regulation and genes associated with cell-to-cell and cell/ECM communications. Co-cultivation of U87-175.4 and U87-Lux8 with HUVE cells demonstrated that U87-175.4 cells suppress the angiogenic phenotype of HUVEC and increase their sensitivity to radiation-induced apoptosis compared to co-culture of U87-Lux8/HUVEC. These data suggest that blockade of p53 function may alter the communication between tumor cells and endothelial cells such that endothelial cells exhibit an increase in radiosensitivity. These findings may have important implications for the treatment of glioblastoma tumors and other human cancers.

Apoptosis↗

PubMed2004

STAT1 is overexpressed in tumors selected for radioresistance and confers protection from radiation in transduced sensitive cells.

Nu61, a radiation-resistant human tumor xenograft, was selected from a parental radiosensitive tumor SCC-61 by eight serial cycles of passage in athymic nude mice and in vivo irradiation. Replicate DNA array experiments identified 52 genes differentially expressed in nu61 tumors compared with SCC-61 tumors. Of these, 19 genes were in the IFN-signaling pathway and moreover, 25 of the 52 genes were inducible by IFN in the nu61 cell line. Among the genes involved in IFN signaling, STAT1alpha and STAT1beta were the most highly overexpressed in nu61 compared to SCC-61. STAT1alpha and STAT1beta cDNAs were cloned and stably transfected into SCC-61 tumor cells. Clones of SCC-61 tumor cells transfected with vectors expressing STAT1alpha and STAT1beta demonstrated radioprotection after exposure to 3 Gy (P < 0.038). The results indicate that radioresistance acquired during radiotherapy treatment may account for some treatment failures and demonstrate an association of acquired tumor radioresistance with up-regulation of components of the IFN-related signaling pathway.

Animals↗

PubMed2003

Tumour-endothelium interactions in co-culture: coordinated changes of gene expression profiles and phenotypic properties of endothelial cells.

Tumour angiogenesis is a complex process based upon a sequence of interactions between tumour cells and endothelial cells. To model tumour/endothelial-cell interactions, we co-cultured U87 human glioma cells with human umbilical vein endothelial cells (HUVECs). U87 cells induced an 'activated' phenotype in HUVECs, including an increase in proliferation, migration and net-like formation. Activation was observed in co-cultures where cells were in direct contact and physically separated, suggesting an important role for soluble factor(s) in the phenotypic and genotypic changes observed. Expressional profiling of tumour-activated endothelial cells was evaluated using cDNA arrays and confirmed by quantitative PCR. Matching pairs of receptors/ligands were found to be coordinately expressed, including TGFbetaRII with TGFbeta3, FGFRII and cysteine-rich fibroblast growth factor receptor (CRF-1) with FGF7 and FGF12, CCR1, CCR3, CCR5 with RANTES and calcitronin receptor-like gene (CALCRL) with adrenomedullin. Consistent with cDNA array data, immunohistochemical staining of expressed proteins revealed the upregulation of Tie-2 receptor in vitro and in vivo. Our data suggest that tumour-induced activation of quiescent endothelial cells involves the expression of angiogenesis-related receptors and the induction of autocrine growth loops. We suggest that tumour cells release growth factors that induce endothelial cells to express specific ligands and their cognate receptors coordinately.

Angiogenesis Inhibitors↗

PubMed2003

Receiver operating characteristic analysis: a general tool for DNA array data filtration and performance estimation.

A critical step for DNA array analysis is data filtration, which can reduce thousands of detected signals to limited sets of genes. Commonly accepted rules for such filtration are still absent. We present a rational approach, based on thresholding of intensities with cutoff levels that are estimated by receiver operating characteristic (ROC) analysis. The technique compares test results with known distributions of positive and negative signals. We apply the method to Atlas cDNA arrays, GeneFilters, and Affymetrix GeneChip. ROC analysis demonstrates similarities in the distribution of false and true positive data for these different systems. We illustrate the estimation of an optimal cutoff level for intensity-based filtration, providing the highest ratio of true to false signals. For GeneChip arrays, we derived filtration thresholds consistent with the reported data based on replicate hybridizations. Intensity-based filtration optimized with ROC combined with other types of filtration (for example, based on significances of differences and/or ratios), should improve DNA array analysis. ROC methodology is also demonstrated for comparison of the performance of different types of arrays, imagers, and analysis software.

Data Interpretation, Statistical↗

PubMed2002

Activation of the nuclear transcription factor kappaB (NFkappaB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine.

PURPOSE: Amifostine has been approved as a therapy to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation treatment for head-and-neck cancer. As a reducing agent capable of participating in intracellular reductive/oxidative processes, it has the potential to affect redox-sensitive transcription factors and gene expression. Amifostine's active free thiol WR-1065 was investigated to determine its effect on nuclear transcription factor kappaB (NFkappaB) activation and subsequent gene expression in U87 glioma cells. METHODS AND MATERIALS: The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 microM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFkappaB activation was determined by a gel shift assay. Changes in gene expression as a function of time of exposure to WR-1065 were determined by Northern blot and the Atlas Human cDNA Expression Array (Clontech, Palo Alto, CA). Changes in gene expression using the Atlas Array were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers. RESULTS: Exposure of U87 cells to 40 microM WR-1065 resulted in a marked activation of NFkappaB between 30 min and 1 h after treatment. Expression of MnSOD, an NFkappaB-responsive gene, was enhanced by over 2-fold after 16 h of treatment and remained elevated at 24 h. During this period of time, no changes in cell cycle distribution were observed. To assess changes in the expression levels of NFkappaB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFkappaB were used. Only five genes were found to be significantly affected at 1, 4, and/or 16 h of treatment. GST-3 and c-myc were repressed up to 2- and 4-fold, respectively. The expression levels of IL-2Ra, RANTES, and c-myb, in contrast, were enhanced up to 14-, 3-, and 2-fold, respectively. The remaining genes having NFkappaB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065. CONCLUSIONS: The redox-sensitive transcription factor NFkappaB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFkappaB by the antioxidant WR-1065 is accompanied by a reduced expression of the oncogene c-myc and an enhanced expression of the antioxidant gene MnSOD, a gene whose expression in tumor cells is relatively low, but when overexpressed has been correlated with a suppression of the malignant phenotype. Activation of NFkappaB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFkappaB-responsive elements.

Amifostine↗