Conserved sequence related to the 3'-terminal region of retrovirus RNA'S In normal cellular DNAs.

The nucleotide sequences related to the 3'-terminal protion of retrovirus genomic RNA have been detected in the DNA of animals, including humans. The DNA complementary to the 400 to 700 nucleotides from the 3'-terminal end of retrovirus RNA (cDNA3'), which contains the enriched conserved region, was hybridized with DNA from a variety of animal cells. Under the conditions of annealing in 0.72 M NaCl at 67 degrees C and hydroxyapatite chromatography at 55 degrees C, 20 to 50% of the radioactivity of the cDNA3' prepared from two retroviruses, a murine Rauscher virus (RLV) and a baboon virus (M7), annealed with normal cellular DNA of animals, including human tissue. The thermal denaturation profile revealed considerable mismatching between the duplex of the cDNA3' and human DNA, cDNA3' of retroviruses is most homologous to cellular DNA of the host species of origin and is less homologous to cellular DNA of species that are distant in the phylogeny of the host species. The conservation and evolution of nucleotide sequences related to the 3' end of retrovirus genomes in animal DNAs, including humans, suggest that the sequences may have important functions.     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Persistent oxidative stress in human colorectal carcinoma, but not in adenoma.

Few studies have been conducted focusing on a potential role of reactive oxygen species in tumor cell metabolism. Here we studied human colorectal adenocarcinomas and adenomas to determine whether oxidative stress is imposed on cancer cells in vivo and used specific antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE)-modified proteins, and 3-nitro-L-tyrosine (3-NT) to determine whether there is an association between oxidative stress and cellular proliferation. Higher levels of oxidative modifications in DNA and proteins were observed in carcinoma cells, but not in adenoma cells, than in the corresponding nontumorous epithelial cells by immunohistochemistry as well as high-performance liquid chromatography (HPLC)-based 8-OHdG determination. The fraction of proliferating cell nuclear antigen-positive cells was proportionally associated in adenocarcinomas with the staining intensities of 8-OHdG and 3-NT. Furthermore, Western blot analysis of the proteins extracted from carcinoma cells revealed several specific proteins modified by HNE or peroxynitrite. Thus we concluded that colorectal carcinoma, but not adenoma cells, are exposed to more oxidative stress than their corresponding nontumorous epithelial cells, regardless of clinical stage and histology, and further that the oxidative stress in carcinoma cells might stimulate cellular proliferation.     

Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium.

Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.     

Quantitative fluorescence imaging approach for the study of polyploidization in hepatocytes.

We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms.     

Primate origin of the cell-derived sequences of simian sarcoma virus

We sought to identify the species of origin of the cell-derived (sis) sequences of simian sarcoma virus. A molecular clone comprised of sis DNA detected related nucleotide sequences at low copy numbers in normal cellular DNAs of species as diverse as humans and quail. The extent of hybridization and degree of base-pair matching with sis DNA were greatest with New World primate DNAs. The thermal denaturation curve midpoints of hybrids formed between sis and woolly monkey DNAs were indistinguishable from homologous sis DNA hybrids, establishing the woolly monkey (Lagothrix spp.) as the source of sis sequences. In comparative studies, sis was shown to be more conserved among mammalian species than unique-sequence woolly monkey cellular DNA. There was no detectable homology between sis and the cell-derived sequences of other fibroblast-transforming retroviruses. These findings indicate that sis is likely to be a unique onc gene among transforming retroviruses.     

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.     

Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans.

We tested the hypothesis that DNA is a target for solar-simulated ultraviolet radiation (ssUVR)-induced suppression of the reactivation of memory immunity in humans. T4N5 liposomes contain the DNA repair enzyme T4 endonuclease V. This cleaves DNA at the site of ultraviolet radiation (UVR)-induced cyclobutane pyrimidine dimers (CPD), initiating DNA repair. It has previously been used to show that CPDs are a key molecular trigger for UVR-induced immunosuppression in mice. To determine whether CPD formation is involved in UVR immunosuppression in humans, nickel-allergic volunteers were irradiated with a range of doses of ssUVR. T4N5 or empty liposomes were then applied after irradiation. Nickel-induced recall immunity was assessed by reflectance spectrometry. T4N5 liposomes inhibited immunosuppression and prevented ssUVR from reducing the number of epidermal dendritic cells. T4N5 liposomes also reduced macrophage infiltration into irradiated epidermis. These studies show that enhanced removal of CPDs from human skin protects from immunosuppression, hence demonstrating that these photolesions are an important molecular event in ssUVR-induced immunosuppression in humans. CPDs also triggered loss of dendritic cells and infiltration by macrophages. It is possible that these changes to antigen presenting cells contribute to ssUVR induced suppression of recall immunity to nickel in humans.     

Arsenicals affect base excision repair by several mechanisms

Inorganic arsenic is a strong, widespread human carcinogen. How exactly inorganic arsenic exerts carcinogenicity in humans is as yet unclear, but it is thought to be closely related to its metabolism. At exposure-relevant concentrations arsenic is neither directly DNA reactive nor mutagenic. Thus, more likely epigenetic and indirect genotoxic effects, among others a modulation of the cellular DNA damage response and DNA repair, are important molecular mechanisms contributing to its carcinogenicity. In the present study, we investigated the impact of arsenic on several base excision repair (BER) key players in cultured human lung cells. For the first time gene expression, protein level and in case of human 8-oxoguanine DNA glycosylase 1 (hOGG1) protein function was examined in one study, comparing inorganic arsenite and its trivalent and pentavalent mono- and dimethylated metabolites, also taking into account their cellular bioavailability. Our data clearly show that arsenite and its metabolites can affect several cellular endpoints related to DNA repair. Thus, cellular OGG activity was most sensitively affected by dimethylarsinic acid (DMA(V)), DNA ligase IIIα (LIGIIIα) protein level by arsenite and X-ray cross complementing protein 1 (XRCC1 protein) content by monomethylarsonic acid (MMA(V)), with significant effects starting at ≥3.2μM cellular arsenic. With respect to MMA(V), to our knowledge these effects are the most sensitive endpoints, related to DNA damage response, that have been identified so far. In contrast to earlier nucleotide excision repair related studies, the trivalent methylated metabolites exerted strong effects on the investigated BER key players only at cytotoxic concentrations. In summary, our data point out that after mixed arsenic species exposure, a realistic scenario after oral inorganic arsenic intake in humans, DNA repair might be affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arsenic.     

Synthesis and biological evaluation of thiobenzanilides as anticancer agents

A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thiobenzanilide agents (4a–c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a–c on human melanoma A375 cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a–c). In addition, DNA flow cytometric analysis shows that 4a–c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in 4a–c-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with 4a–c resulted in the loss of mitochondrial membrane potential (ΔΨ_mt), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a–c agents are potent inducers of cell apoptosis in A375 cells.     

Catalase ameliorates polychlorinated biphenyl-induced cytotoxicity in nonmalignant human breast epithelial cells

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human nonmalignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), significantly decreased cell number and MTS reduction and increased the percentage of cells with sub-G1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pretreated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.     

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.     

A quantitative evaluation of cytophotometric DNA analysis in retrospective studies using archival tumor specimens.

Methods developed for the cytophotometric analysis of archival tumor specimens used in retrospective studies were evaluated quantitatively. May-Grünwald-Giemsa-stained cytologic slides up to 20 years old could be restained by the Feulgen reaction with excellent results if they were destained in methanol and refixed in formaldehyde prior to Feulgen staining. Storage time had only a minor influence on Feulgen stainability. However, a considerable variation in the intensity of the Feulgen stain was observed between different slides stained simultaneously; this variation was not related to storage time. As a consequence of this variation, the use of internal staining controls, such as granulocytes, is an absolute necessity in the quantitative comparison of different slides. By expressing DNA data from the tumor cells in relative values (c values) related to the internal staining control (with a defined mean value of 2c), Feulgen ploidy level determinations could be made as accurately from measurements on old, destained slides as on cells obtained from fresh tumor material. The ploidy level could also be accurately determined in most cases of prostatic carcinoma from measurements on histopathologic sections.     

Review of the genotoxicity of ozone.

Ozone is a powerful oxidant, reactive to biomolecules. In aqueous solution it decomposes to give hydrogen peroxide, superoxide and hydroxy radicals which can take part in secondary reactions. Ozone is a disinfectant that inactivates both viruses and bacteria. Although other reactions are primarily responsible for the inactivation, cellular DNA is also damaged. Ozone is genotoxic to microorganisms, plants and cell cultures in vitro. The results from in vivo cytogenetic studies with laboratory animals after inhalation exposure are contradictory. Chromosome aberrations in lymphocytes, but not SCEs, have been demonstrated in Chinese hamsters but not in mice. Chromatid deletions were induced in pulmonary macrophages in rats. No cytogenetic effects have been reported for bone marrow cells or spermatocytes. The few experimental and epidemiological studies with human subjects do not allow a conclusion on the cytogenetic effects of ozone in lymphocytes in humans. No life-long cancer studies have been performed with ozone. However, after 4 and 6 months of inhalation exposure, lung adenomas were induced in strain A/J mice, but not in Swiss-Webster mice.     

Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present and Future. R. Stanyon M. Rocchi(b) F. Bigoni(a) N. Archidiacono(b)

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.     

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a non-toxic concentrations (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.     

Replication of human cytomegalovirus DNA: lack of dependence on cell DNA synthesis.

Human cytomegalovirus (CMV) DNA synthesis was studied in 5-fluorouracil (FU)-treated and untreated human embryonic lung cells, which differ greatly with respect to the number of cells in the culture synthesizing cellular DNA. CMV DNA synthesis proceeded at the same rate in FU-treated and in untreated cells. CMV infection also reversed the inhibitory effects of FU and activated cellular DNA synthesis in some of the cells in the FU-treated culture. Autoradiographic studies showed that more than 20% of the cells in the infected FU-treated culture synthesized viral DNA when less than 1% had synthesized cellular DNA, indicating that the synthesis of viral macromolecules proceeds in cells that do not synthesize cellular DNA from the time of infection, and that viral DNA synthesis proceeds independently of the host cell DNA synthesis. Combined autoradiographic and immunofluorescence studies of both the FU-treated and untreated infected cells showed that, whereas 20% of the cells in the cultures synthesize viral DNA and viral antigens, only about 3 to 6% of those cells that synthesize cellular DNA also synthesize viral antigen. Thus, productive infection was delayed or inhibited in those cells that were stimulated by CMV infection to synthesize cellular DNA.     

Radioautographic and cytofluorimetric analysis of DNA synthesis during the pre-implantation period of rat and mouse embryonic development]

The patterns of DNA synthesis and kinetics of cell population in the rat and mouse embryos were studied by means of 3H-thymidine autoradiography and cytofluorimetry. The rat and mouse embryos during the period of cleavage consist of a heterogenous population of blastomeres. At all the stages under study, all phases of the cell cycle occur in the blastomeres: G1, S, G2 and mitosis. The embryonic cells were distributed into groups containing 2c, 3c, 4c and more DNA. The ratio of cell number in these groups differed in the mouse and rat embryos. The mouse embryos are characterized by the appearance of a considerable amount of polyploid cells in S phase at the morula stage. The stage and species specific quantitative and qualitative patterns were established for DNA synthesis and kinetics of the cell population of blastomeres.