Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.     

Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis

Isoetes coreana Chung and Choi is on the list of critically endangered species in South Korea. Using randomly amplified polymorphic DNA (RAPD) markers, we investigated the genetic diversity within and between seven local populations of I. coreana. Ten RAPD primers produced a total of 94 bands, of which 59 (62.8%) were polymorphic. A low level of genetic diversity was recognized within the populations of I. coreana: polymorphic loci (P), with values ranging from 3.4% to 33.9%, and a mean value of 15.5% being observed. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater among populations (81.6%) than within populations (19.7%) or among the three regions included in this study (Han River, Youngsan River, and Nakdong River). In addition, a high degree of genetic differentiation (θ^B = 0.742) was detected among the populations. These results indicate that genetic differentiation has occurred very rapidly. However, the rate of gene flow between populations was found to be as low as 0.059, irrespective of the genetic and geographical distances between the populations, which indicates that genetic drift must have played an important role in forming the present populations of I. coreana. Because a reduction of genetic diversity as a result of genetic drift is undesirable, increasing the gene flow between populations of Korean quillwort I. coreana should be considered as a conservation strategy.     

Observation and control in a model of a cell population affected by radiation

The effect of radiation on a cell population is described by a two-dimensional nonlinear system of differential equations. If the radiation rate is not too high, the system is known to have an asymptotically stable equilibrium. First, for the monitoring of this effect, the concept of observability is applied. For the case when the total number of cells is observed, without distinction between healthy and affected cells, a so-called observer system is constructed, which, at least near the equilibrium state, makes it possible to recover the dynamics of both the healthy and the affected cells, from the observation of the total number of cells without distinction.     

Genetic diversity and population structure of Taxus cuspidata in the Changbai Mountains assessed by chloroplast DNA sequences and microsatellite markers

Taxus cuspidata, a tree species with high economic value because of its anticancer properties, is experiencing severe reduction in populations across its range in China. We examined one chloroplast DNA (cpDNA) region (petA-psbE) and 9 nuclear simple sequence repeats (SSRs) loci variations among seven populations in the Changbai Mountains of China to investigate the levels of genetic diversity and population structure. A moderate level of haplotype diversity (H_T = 0.625), low nuclear microsatellite diversity (H_E = 0.261 ± 0.028), significant genetic differences (F_ST = 0.065) and substantial gene flow (Nm = 2.806) were observed. Most of the total genetic variation was partitioned within the population (87.8% and 94.0% for cpDNA and SSRs, respectively). Our haplotype identification permutation tests revealed that G_ST > N_ST, indicating an absence of phylogeographic structure in T. cuspidata. Neither STRUCTURE nor UPGMA analyses showed any geographic pattern in T. cuspidata populations. By comparatively analyzing the genetic diversity and survival situation of T. cuspidata, our results provide a theoretical foundation for the resource protection, utilization cultivation and breeding of this valuable plant.     

Cell line studies and analytical measurements of three paclitaxel complex variations

The copper(II) cation, sucrose, and hydroxychloroquine were complexed with the chemotherapy agent paclitaxel and studied for medicinal activity. Data (GI₅₀, LD₅₀) from single dose and five dose National Cancer Institute sixty cell line panels are presented. Analytical measurements of different complexes were made using Nuclear Magnetic Resonance (¹H NMR), Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) and Fourier Transform-Ion Cyclotron Resonance (FT-ICR). Molecular modeling is utilized to better understand the impact that species could have on physical parameters associated with Lipinski’s Rule of Five, such as log P and TPSA. On average, Cu(II) and hydroxychloroquine decreased GI₅₀ values, while sucrose increased GI₅₀ values of paclitaxel.     

一株新的人肾癌原代细胞系的建立及其生物学特性鉴定

目的:取肾癌病人标本建立一株新的人肾癌细胞系,初步对该细胞系进行鉴定,为进一步的肾癌基础研究提供实验模型.方法:2008-2009年间共采集20例肾癌新鲜手术标本,于手术后1 h内将每例标本切取四块0.5cm× 0.5 cm× 0.5 cm组织块,分别包埋于2只裸鼠的右后肢皮下及背部皮下,连续传代3次,取移植瘤体外培养,记录细胞株的生长曲线,测定细胞集落形成率,对细胞进行DNA含量测定,进行染色体分析及病理学检查.结果:其中1只裸鼠皮下移植瘤生长,继续传代,肿瘤生长速度明显加快.取移植瘤标本体外培养得到肾癌细胞系XJG-9201.形态结构,分化程度与原发瘤一致,染色体众数为65.细胞群体倍增时间为38.2h,细胞周期分析G1期62.7%,G2期11.2%,S1期25.3%,集落形成率为70%.结论:肾癌细胞系XJG-9201与原发癌保持相同的生物学特性,体外连续传代1年以上传115代.细胞形态不变,生长周期恒定,已成为一个稳定的细胞系.     

Damaging and protective cell signalling in the untargeted effects of ionizing radiation

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.     

Genetic variability in a genomic region with long-range linkage disequilibrium reveals traces of a bottleneck in the history of the European population

The inference of the demographic history of populations from genetic variability data is not only of academic interest. It also provides background information for the identification of genes which may have played a role in human evolution or in the aetiology of human disease. To obtain a clear picture of this background, it is necessary to compare data obtained from a number of genomic loci. Due to its very low recombination rate, the NF1 gene region can be regarded as a further suitable locus. A combined resequencing and SNP typing project in a European population disclosed the presence of only two well separated subgroups of NF1 sequences. Statistical analysis revealed a bimodal distribution of the pairwise differences, a positive value of Tajima’s D and a TMRCA of 700,000 years for the whole sample, and pairwise differences indicative for a growing population and TMRCAs of 130,000 to 150,000 years for the subgroups. Together, the data lead to a model that the recent European population went through a bottleneck during the last 150,000 years of its history. Regarding the given timeframe, this bottleneck could either reflect a speciation event which led to the anatomically modern human (AMH), or a severe reduction of the population size during the emigration of AMHs out of Africa or the immigration into Europe.     

Genetic differentiation of Pseudoregma bambucicola population based on mtDNA COII gene

mtDNA COII gene sequences were identified and analyzed using different types of software, namely, MEGA5.0, DNAMAN, and DnaSP5.0 in four Chinese provinces, namely, Sichuan, Zhejiang, Guizhou and Shanghai. Analysis of molecular genetic variation and its genetic structure and differentiation, combined with NJ tree, MP tree analysis and analysis of molecular variance (AMOVA), at Fst = 0.0582 conclude that the genetic differentiation is low, gene flow is Nm = 8.0911, and gene exchange is sufficient. However, for the geographic populations of Pseudoregma bambucicola in the four provinces, their gene exchange is relatively weak at Nm = 0.8284, whereas the genetic differentiation is high at Fst = 0.3764. Based on the data, total nucleotide diversity between the populations is 0.00158 ± 0.00021. The results showed that the total population of Tajima’s D and Fu’s Fs results are D = ?0.885 and Fs = 0.226, respectively. The experimental numerical results showed that this total population is not significant (P > 0.10), indicating that nine different geographic populations are short-term. No expansion occurred in the internal population. This study provided a theoretical and practical basis for the comprehensive prevention and control of P. bambucicola.     

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

Inhibition of C-terminal truncated PPM1D enhances the effect of doxorubicin on cell viability in human colorectal carcinoma cell line

PPM1D is a p53-inducible Ser/Thr phosphatase. One of the main functions of PPM1D in normal cells is to act as a negative regulator of the p53 tumor suppressor by dephosphorylating p53 and several kinases. PPM1D is considered an oncoprotein owing to both its functions and the fact that gene amplification and overexpression of PPM1D are reported in several tumors. Recently, PPM1D mutations resulting in C-terminal truncated alterations were found in brainstem gliomas and colorectal cancers, and these mutations enhanced the activity of PPM1D. Therefore, C-terminal truncated PPM1D should be also considered as a potential candidate target of anticancer drugs. Here we showed that combination treatment with PPM1D-specific inhibitor SPI-001 and doxorubicin suppressed cell viability of HCT-116 cells overexpressing C-terminal truncated PPM1D through p53 activation compared with doxorubicin alone. Our results suggest that combination treatment with PPM1D inhibitor and doxorubicin may be a potential anti-cancer treatment in PPM1D-mutated cancer cells.     

Genetic diversity patterns in Phragmites australis at the population,regional and continental scales

Genetic diversity, population structure and interrelationships were investigated in eight populations of the common reed, Phragmites australis, in the Po Plain, Italy, by means of amplified fragments length polymorphisms (AFLPs) and random amplified polymorphic DNAs (RAPDs). Patterns of genetic diversity were analysed in relation to size, age and degree of human impact in the wetlands and compared with that of a distant population in Romania. Genetic distances between Po Plain clones and geographically distant clones were measured to determine the geographical extent of the gene pool.     

Genetic structure of the Eurasian lynx population in north-eastern Poland and the Baltic states

We analyzed the genotypes of Eurasian lynx (Lynx lynx) from three populations in the westernmost part of the species main range. One population was situated at the distribution edge (NE Poland) and the two other (Latvia and Estonia) were located within the main, contiguous range of the species. The aim was to determine if the genetic composition varied among these populations and if there was evidence of isolation among them. Based on microsatellite allele frequencies, we found the allelic richness in Polish lynx to be lower than that in lynx from Latvia and Estonia. We also found significant differentiation among the lynx populations, with the NE Poland population forming a distinct genetic group relative to the two other populations (R _ST = 0.15 and 0.22, P < 0.0001). We suggest that genetic differentiation among lynx populations is the result of habitat insularisation that limits gene flow. This finding emphasizes the necessity to consider the lynx genetic differentiation in conservation planning of this species in Poland.     

Histogenesis of carcinosarcoma and Establishment of leiomyosarcoma cell line (HTMMT) derived from human uterine carcinosarcoma

A cell line designated HTMMT was established from the human uterine carcinosarcoma (composed of leiomyosarcoma and adenocarcinoma) of a 66-year-old Japanese woman. The cell line grew well and 83 serial passages were successively done within 24 months. The cell line contained spindle- or fibrous-shaped cells that revealed neoplastic and pleomorphic features, and multipled rapidly without contact inhibition. These cells were characterized as possessing myofibrils. The karyotype exhibited hyperploidy and the chromosome number was ranged from 87 to 100. The cells were transplanted into an immune-depressed hamster cheek pouch or into nude mouse skin, but produced no tumors. We supported the combination theory for the histogenesis of the carcinosarcoma.     

Establishment and characterization of new B-cell precursor leukemia cell line NALM-35

A novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) cell line, NALM-35, was established from the peripheral blood of a 40-year-old woman at diagnosis of ALL. Imunophenotyping showed BCP type III characteristics including expression of TdT, CD10, CD19, CD22, CD79a and HLA class II. T-cell and myeloid-associated antigens tested were negative except CD5 and CD28. The surrogate light chains CD179a and CD179b were positive. NALM-35 cells have the morphological appearance of lymphoblasts. Cytogenetic analysis of NALM-35 revealed an abnormal karyotype with 46, XX, add(9)(p11). Southern blot analysis of the immunoglobulin genes status of NALM-35 at 10 months after establishment showed germ line configuration of the kappa and lambda light chain genes, and rearrangement of the mu heavy chain gene. DNA fingerprinting, chromosomal analysis and immunophenotyping proved that NALM-35 was clonally derived from the primary leukemia cells. The established cell line may provide a useful model system and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.     

Genetic neighbourhood structure in a population of Picea abies L.

Summary Natural populations are currently the basic material for studying forest tree breeding, but little is known on the genotypic spatial structures in these stands. The use of gene markers, such as isozymes, leads to the determination of part of the allelic constitution of individuals. A method is presented here to estimate the degree of genetic relationship between any pair of genotypes. A French Picea abies population is analyzed by these means and a slight but significant correlation between estimated genetic relationship and topographic distance is found.     

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures.The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure.     

Brain stem cells as the cell of origin in glioma

Glioma incidence rates in the United States are near 20000 new cases per year, with a median survival time of 14.6 mo for high-grade gliomas due to limited therapeutic options. The origins of these tumors and their many subtypes remain a matter of investigation. Evidence from mouse models of glioma and human clinical data have provided clues about the cell types and initiating oncogenic mutations that drive gliomagenesis, a topic we review here. There has been mixed evidence as to whether or not the cells of origin are neural stem cells, progenitor cells or differentiated progeny. Many of the existing murine models target cell populations defined by lineage-specific promoters or employ lineagetracing methods to track the potential cells of origin. Our ability to target specific cell populations will likely increase concurrently with the knowledge gleaned from an understanding of neurogenesis in the adult brain. The cell of origin is one variable in tumorigenesis, as oncogenes or tumor suppressor genes may differentially transform the neuroglial cell types. Knowledge of key driver mutations and susceptible cell types will allow us to understand cancer biology from a developmental standpoint and enable early interventional strategies and biomarker discovery.     

Establishment and characterization of a lymphoepithelial-like carcinoma cell line (HUUCLEC) derived from the human uterine cervix

A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.     

Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gammaglutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.