Biomedical subjects

Trond Stokke

Publications and source records attributed to Trond Stokke.

At least 19 recordsLinked to original sources

PubMed2006

Gene expressions and copy numbers associated with metastatic phenotypes of uterine cervical cancer.

BACKGROUND: A better understanding of the development of metastatic disease and the identification of molecular markers for cancer spread would be useful for the design of improved treatment strategies. This study was conducted to identify gene expressions associated with metastatic phenotypes of locally advanced cervical carcinomas and investigate whether gains or losses of these genes could play a role in regulation of the transcripts. Gene expressions and copy number changes were determined in primary tumors from 29 patients with and 19 without diagnosed lymph node metastases by use of cDNA and genomic microarray techniques, respectively. RESULTS: Thirty-one genes that differed in expression between the node positive and negative tumors were identified. Expressions of eight of these genes (MRPL11, CKS2, PDK2, MRPS23, MSN, TBX3, KLF3, LSM3) correlated with progression free survival in univariate analysis and were therefore more strongly associated with metastatic phenotypes than the others. Immunohistochemistry data of CKS2 and MSN showed similar relationships to survival. The prognostic genes clustered into two groups, suggesting two major metastatic phenotypes. One group was associated with rapid proliferation, oxidative phosphorylation, invasiveness, and tumor size (MRPS23, MRPL11, CKS2, LSM3, TBX3, MSN) and another with hypoxia tolerance, anaerobic metabolism, and high lactate content (PDK2, KLF3). Multivariate analysis identified tumor volume and PDK2 expression as independent prognostic variables. Gene copy number changes of the differentially expressed genes were not frequent, but correlated with the expression level for seven genes, including MRPS23, MSN, and LSM3. CONCLUSION: Gene expressions associated with known metastatic phenotypes of cervical cancers were identified. Our findings may indicate molecular mechanisms underlying development of these phenotypes and be useful as markers of cancer spread. Gains or losses of the genes may be involved in development of the metastatic phenotypes in some cases, but other mechanisms for transcriptional regulation are probably important in the majority of tumors.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2006

Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery.

BACKGROUND: Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure. RESULTS: 5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G2(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C). CONCLUSION: Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies.

Antimetabolites, Antineoplastic↗

PubMed2006

CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization.

We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP-L, with a 294 amino acids longer N-terminus and a 51 amino acids insertion located in the coiled-coil mid-domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP-L expression is highest in G2/M. Ectopic expression of CSPP-L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP-L but not in CSPP transfectants. CSPP-L relocates from spindle microtubules and poles in metaphase to the mid-spindle in anaphase and concentrates at the mid-body in telophase/cytokinesis. CSPP-L high-expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP-L on microtubule organization in mitosis depend on the coiled-coil mid-domain insertion. The common C-terminal domain is required to repress that activity until mitosis. Notably, this C-terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP-L interact with centrosomes and microtubules and can differently affect microtubule organization.

Cell Cycle↗

PubMed2006

Induction of apoptosis by hexaminolevulinate-mediated photodynamic therapy in human colon carcinoma cell line 320DM.

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.

Aminolevulinic Acid↗

PubMed2005

Bystander effects in UV-induced genomic instability: antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation.

BACKGROUND: Genomic instability is characteristic of many types of human cancer. Recently, we reported that ultraviolet radiation induced elevated mutation rates and chromosomal instability for many cell generations after ultraviolet irradiation. The increased mutation rates of unstable cells may allow them to accumulate aberrations that subsequently lead to cancer. Ultraviolet A radiation, which primarily acts by oxidative stress, and ultraviolet B radiation, which initially acts by absorption in DNA and direct damage to DNA, both produced genomically unstable cell clones. In this study, we have determined the effect of antioxidants on induction of delayed mutations by ultraviolet radiation. Delayed mutations are indicative of genomic instability. METHODS: Delayed mutations in the hypoxanthine phosphoribosyl transferase (hprt) gene were detected by incubating the cells in medium selectively killing hprt mutants for 8 days after irradiation, followed by a 5 day period in normal medium before determining mutation frequencies. RESULTS: The UVB-induced delayed hprt mutations were strongly inhibited by the antioxidants catalase, reduced glutathione and superoxide dismutase, while only reduced glutathione had a significant effect on UVA-induced delayed mutations. Treatment with antioxidants had only minor effects on early mutation frequencies, except that reduced glutathione decreased the UVB-induced early mutation frequency by 24%. Incubation with reduced glutathione was shown to significantly increase the intracellular amount of reduced glutathione. CONCLUSION: The strong effects of these antioxidants indicate that genomic instability, which is induced by the fundamentally different ultraviolet A and ultraviolet B radiation, is mediated by reactive oxygen species, including hydrogen peroxide and downstream products. However, cells take up neither catalase nor SOD, while incubation with glutathione resulted in increased intracellular levels of glutathione. Previously, we have shown that ultraviolet induced delayed mutations may be induced via a bystander effect and that this effect is 5-fold higher for UVB radiation than for UVA radiation. Therefore, we propose that the antioxidants inhibit an ultraviolet radiation-induced bystander effect and that the effect is transmitted via the medium and via an internal transfer between cells, like gap junctional intercellular communication, for UVB radiation and only by the latter mechanism for UVA radiation.

Journal Article↗

PubMed2005

Response of malignant B lymphocytes to ionizing radiation: gene expression and genotype.

The human malignant B-lymphocyte cell lines Reh and U698 show arrest in G2 phase after ionizing radiation (IR), but only Reh cells arrest in G1 phase and die by apoptosis. We have used cDNA microarrays to measure changes in gene expression at 2, 4 and 6 hr after irradiation of Reh and U698 cells with 0.5 and 4 Gy in order to begin exploring the molecular mechanisms underlying the phenotypic changes. We also investigated whether gene expression changes could be caused by possible aberrations of genes, as measured by comparative genomic hybridization. Reh cells showed upregulation of CDKN1A that likely mediated the G1 arrest. In contrast, U698 cells have impaired function of TP53 protein and no activation of CDKN1A, suppressing the arrest in G1. The G2 arrest in both cell lines was likely due to repression of PLK1 and/or CCNF. IR-induced apoptosis in Reh cells was probably mediated by TP53 and CDKN1A, whereas a high expression level of MCL1, caused by gene amplification, and activation of the NFKB pathway may have suppressed the apoptotic response in U698 cells. Genes suggested to be involved in apoptosis were activated long before this phenotype was detectable and showed the same temporal expression profiles as genes involved in cell cycle arrest. Our results suggest that differences in functionality and/or copy number of several genes involved in IR-regulated pathways contributed to the phenotypic differences between Reh and U698 cells after IR, and that multiple molecular factors control the radiation response of malignant B lymphocytes.

Chromosome Aberrations↗

PubMed2005

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization.

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G(1)/S-phase progression and spindle assembly.

Amino Acid Sequence↗

PubMed2005

Bystander effects may modulate ultraviolet A and B radiation-induced delayed mutagenesis.

Ultraviolet irradiation of cells can induce a state of genomic instability that can persist for several cell generations after irradiation. However, questions regarding the time course of formation, relative abundance for different types of ultraviolet radiation, and mechanism of induction of delayed mutations remain to be answered. In this paper, we have tried to address these questions using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay in V79 Chinese hamster cells irradiated with ultraviolet A or B radiation. Delayed HPRT(-) mutations, which are indications of genomic instability, were detected by incubating the cells in medium containing aminopterin, selectively killing HPRT(-) mutants, and then treating the cells with medium containing 6-thioguanine, which selectively killed non-mutant cells. Remarkably, the delayed mutation frequencies found here were much higher than reported previously using a cloning method. Cloning of cells immediately after irradiation prevents contact between individual cell clones. In contrast, with the present method, the cells are in contact and are mixed several times during the experiment. Thus the higher delayed mutation frequency measured by the present method may be explained by a bystander effect. This hypothesis is supported by an experiment with an inhibitor of gap junctional intercellular communication, which reduced the delayed mutation frequency. In conclusion, the results suggest that a bystander effect is involved in ultraviolet-radiation-induced genomic instability and that it may be mediated in part by gap junctional intercellular communication.

Aminopterin↗

PubMed2004

Intratumor chromosomal heterogeneity in advanced carcinomas of the uterine cervix.

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2-4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14-q25 (60% of 10 cases with this aberration), and gain of 2p22-pter (50% of 6 cases), 11qcen-q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and -X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages.

Biopsy↗

PubMed2004

Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction.

BACKGROUND: High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure. RESULTS: All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range. CONCLUSIONS: It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Algorithms↗

PubMed2004

Inhibitory targeting of checkpoint kinase signaling overrides radiation-induced cell cycle gene regulation: a therapeutic strategy in tumor cell radiosensitization?

BACKGROUND AND PURPOSE: The tumor cell defense response to ionizing radiation involves a temporary arrest at the cell cycle G(2) checkpoint, which is activated by a signaling cascade initiated by the ATM kinase response to DNA damage, ultimately leading to the outcome of further cell survival if the DNA is properly repaired. The inhibitory targeting of the checkpoint kinase signaling elicited by ATM may define a biologically based strategy to override the G(2) phase delay that prevents mitotic entry after DNA damage, thereby increasing the probability of mitotic cell death following exposure to ionizing radiation. MATERIALS AND METHODS: Breast carcinoma cell lines with intact or defective function of the tumor-suppressor protein BRCA1 were exposed to ionizing radiation in the absence or presence of a specific inhibitor (UCN-01) of the checkpoint kinase CHK1, and the response profiles of cell cycle distribution and G(2) phase regulatory factors, as well as the efficiency of clonogenic regrowth, were analyzed. RESULTS: The radiation-induced G(2) phase accumulation was preceded by a transient down-regulation of the G(2) phase-specific polo-like kinase-1 and cyclin B1, which required intact function of both BRCA1 and CHK1. The concomitant treatment with UCN-01 seemed to amplify the cytotoxic effect of ionizing radiation on clonogenic regrowth. CONCLUSION: The effector mechanism of DNA damage on cell cycle gene regulation signals through the checkpoint kinase network. Among molecular cell cycle-targeted drugs currently in pipeline for testing in early phase clinical trials, CHK1 inhibitors may have therapeutic potential as radiosensitizers.

Animals↗

PubMed2004

The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole.

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.

Apoptosis↗

PubMed2004

Molecular characterizations of derivatives of HCT116 colorectal cancer cells that are resistant to the chemotherapeutic agent 5-fluorouracil.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

Antimetabolites, Antineoplastic↗

PubMed2003

Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1.

DNA damage activates the G2 cell cycle checkpoint to allow time for DNA repair before mitotic entry. The mechanism involves inhibition of the enzymatic activity for polo-like kinase 1 (Plk1), rendering Cdc25C with a basal phosphatase activity that is insufficient for converting Cdc2 to the fully active G2/M transition kinase. We found that cell cycle arrest at the G2/M boundary after ionizing radiation (IR) of breast carcinoma cells may involve repression of the gene for Plk1, PLK, mediated by the tumor-suppressor protein BRCA1. The p53-defective MT-1 cell line had an apparent accumulation of G2/M phase cells 12 h after irradiation. This response was preceded by a transient downregulation of PLK mRNA expression with a barely detectable level 6 h after exposure to IR but recovered after 12 h. A significantly lower fraction of irradiated BRCA1(-/-) HCC1937 cells arrested in the G2/M phase after 12 h, and the transient response of PLK mRNA was also considerably impaired. After reconstitution of wild-type BRCA1 in the HCC1937 cells however, downregulation of PLK mRNA as well as Plk1 protein expression after IR was restored. Moreover, the suppression of PLK mRNA expression 6 h after irradiation was completely abolished by the specific CHEK1 kinase inhibitor UCN-01, further indicating that the effector mechanism of DNA damage on PLK signals through BRCA1 and its downstream CHEK1. Our observations provide new information about the diversity of regulatory mechanisms governed by BRCA1 in DNA damage checkpoint control.

BRCA1 Protein↗

PubMed2003

Photochemically enhanced gene delivery with cationic lipid formulations.

Entrapment and degradation of transfecting DNA in endocytic vesicles often hampers the use of lipidic vectors for gene delivery purposes. Photochemical internalisation (PCI) is a technology for achieving light-induced release of DNA trapped inside these vesicles, and therefore represents a way of overcoming the endocytic membrane barrier and improving gene transfer. The technology is based on utilising photosensitizers which localise in the membranes of endocytic vesicles, causing photochemical damages that rupture the vesicles upon illumination. The purpose of this work was to study the effect of PCI on transfection mediated by the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (betaAE-DMRIE), with or without the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). It was shown that PCI has no effect on betaAE-DMRIE mediated transfection, whereas it significantly enhances transfection mediated by the combination of betaAE-DMRIE and DOPE. The effect of PCI was highly dependent on the timing of illumination relative to the time of DNA delivery, both regarding the sequence of, and the time between, these two treatments.

Cations↗

PubMed2002

The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.

BACKGROUND: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival. METHODS: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index. RESULTS: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators. CONCLUSIONS: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

Antibiotics, Antineoplastic↗

PubMed2002

Dye exclusion artifact in flow cytometers.

BACKGROUND: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. MATERIALS AND METHODS: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. RESULTS: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. CONCLUSIONS: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.

Animals↗

PubMed2002

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells.

OBJECTIVE: Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS: The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS: Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.

Adult↗