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Biomedical subjects

H Werbin

Publications and source records attributed to H Werbin.

At least 19 recordsLinked to original sources
PubMed1997

Telomerase assays in the diagnosis and prognosis of cancer.

Telomerase activity is present in most primary human tumours but not in normal somatic tissues except for proliferative cells of renewal tissues (e.g. crypts of the intestine, basal layer of the epidermis, haemopoietic and inflammatory cells). In some instances telomerase activity is detected in preinvasive lesions, whereas in others it is only detected at later stages. Lower telomerase activity levels are detected in some specimens obtained from regions adjacent to primary tumours. The key clinical challenge is to determine if the presence or level of telomerase activity has diagnostic or prognostic utility. Almost any clinical specimen can be used to assay telomerase activity including frozen sections, fine needle aspirates, brushes, washes and sedimented cells in voided urine. In certain cancers increased telomerase activity levels may identify patients that will have either favourable or poor prognostic outcomes, whereas in other instances telomerase activity can distinguish between benign and malignant lesions. New approaches to improve the diagnostic value of telomerase determinations include application of in situ hybridization methods for detecting human telomerase RNA expression on archival paraffin-embedded material. Results show that this assay easily distinguishes cancer from normal cells, and thus may complement the telomerase activity assays.

Biomarkers, Tumor↗
PubMed1996

Telomeres and telomerase in human leukemias.

There is increasing evidence supporting the hypothesis that telomere shortening both in vitro and in vivo, is the clock that counts cell divisions and determines the onset of cellular senescence. Cells that overcome the normal senescence mechanisms do so by stabilizing telomere length, probably due to the activity of telomerase, a ribonucleoprotein enzyme that synthesizes telomeric repeats. Most human primary tumors contain telomerase, while the cells of most normal tissues lack this activity. A hypothesis gaining prominence is that the activation of telomerase is necessary for the sustained growth of most solid tumors. Since normal hematopoietic stem cells and some of their progeny already express telomerase activity, it is important to consider whether or not telomere shortening and telomerase activity play any role in cancer progression in various forms of leukemia. This review includes a discussion of the utility of telomere length and/or telomerase activity measurements in the diagnosis and prognosis of leukemia as well as the potential value of antitelomerase therapy for the leukemias.

Base Sequence↗
PubMed1993

Toward a molecular understanding of human breast cancer: a hypothesis.

A rate limiting step in most metastatic breast cancers is the development of unlimited proliferative potential by mammary epithelial cells. We describe mechanisms by which these cells can attain this state. The two independent mortality mechanisms controlling fibroblast senescence and immortalization (M1 and M2) are also found in human mammary epithelial cells. However, although both p53 and Rb are involved in the M1 mechanism of fibroblast cellular senescence, in human mammary epithelial cells only p53 is involved. The M1/M2 mechanisms may be induced by the gradual loss of telomere ends that occur as normal cells divide. Loss of telomere ends may result in genomic instability and in altered gene expression due to heterochromatin changes in subtelomeric regions. Events which can abrogate p53 functions are described, as is the current state of knowledge about the function of p53. All these factors are included in a molecular model for the onset of breast cancer.

Breast Neoplasms↗
PubMed1992

Cellular and molecular advances in elucidating p53 function.

The finding that in many human tumors there is allelic loss and/or mutations in p53, in combination with recognition that these events may play a role in multi-stage carcinogenesis, has focused considerable interest on this gene. To help keep abreast of this rapidly expanding field, recent experiments on the role and potential regulation of p53 are described: these include discussions of p53 as an anti-proliferative agent, the p53 mutations found in human tumors and tumor cell lines, the conformational states of p53, phosphorylation of p53 by p34cdc2, and signals for the nuclear localization of p53. p53 may act as a transcriptional activator and the specific DNA sequences to which p53 protein binds are also discussed as is the importance of abrogation of p53 function in overcoming cellular senescence.

Base Sequence↗
PubMed1992

New evidence for the insertion of mitochondrial DNA into the human genome: significance for cancer and aging.

We have observed and characterized in detail two cases of mitochondrial DNA fragments which have inserted into the nucleus of HeLa cells. In one case three non-sequential but contiguous regions of mitochondrial DNA with 92% homology to human cytoplasmic mitochondrial DNA inserted into the nuclear genome. In the second case the mitochondrial DNA sequence encoding cytochrome c oxidase subunit III was contiguous with and 5' of exons 2 and 3 of the c-myc oncogene and the chimeric gene was transcribed. Models are presented that describe mechanisms for the transfer of mitochondrial DNA into the nucleus involving fragmentation of mitochondrial DNA through aging and/or oxidative damage, anomalous processing or escape of mitochondrial DNA and RNA fragments from autophagic vacuoles, and insertion of mitochondrial DNA sequences, in some instances after reverse transcription of mitochondrial RNA, into the nuclear genome.

Adult↗
PubMed1991

Defining the molecular mechanisms of human cell immortalization.

Although the immortalization of human cells is a key step in oncogenic progression, the molecular mechanisms underlying this event are poorly understood. After reviewing the use of chemicals, physical agents, oncogenes and DNA tumor viruses as immortalizing agents, we consider the importance of negative regulators of cell growth (RB and p53), their inactivation, as well as genomic instability in the pathogenesis of cancer. Finally, a molecular model for human cell immortalization that integrates many of the above observations is presented along with supporting evidence.

Cell Transformation, Neoplastic↗
PubMed1990

Mitochondrial DNA copy number is proportional to total cell DNA under a variety of growth conditions.

Analyses of populations of NIH/3T3 cells for mitochondrial DNA under various conditions were made by use of an improved procedure. Cells were examined at different cell densities, at different stages of cell growth, and after the cells had been incubated with a carcinogen reported to affect mitochondrial DNA replication, benzo[a]pyrene-epoxide. In addition, the analysis was performed on three tumorigenic NIH/3T3 cell lines and one mouse bladder epithelial cell line and its tumorigenic derivative. Small variations of mitochondrial DNA under these various conditions were detected easily and revealed a linear relationship between mitochondrial DNA and total cell DNA.

Animals↗
PubMed1990

An improved procedure for quantitating mitochondrial DNA in cultured mammalian cells.

A new improved method that reproducibly measures small perturbations of mitochondrial DNA in populations of cells has been developed. It is based on first obtaining a cell count and then analyzing three aliquots of cells: one for total DNA per cell by fluorometry, one for total protein per cell and one for the amount of mitochondrial DNA per microgram of total cell DNA. To quantitate mitochondrial DNA, 0, 1, 2, and 3 nanograms of mouse mtDNA purified from a plasmid are added as internal standard DNA to four 1.0-microgram samples of purified total cell DNA containing an unknown amount of mitochondrial DNA (a sample set). Three sample sets are electrophoresed in an agarose gel devoid of ethidium bromide. Following Southern transfer to nitrocellulose and hybridization to purified 32P-labeled mouse mitochondrial DNA, an autoradiogram is prepared for use as a template to locate the mitochondrial DNA bands. These bands are cut out of the nitrocellulose filters, and their 32P-content is determined using a liquid scintillation counter. For each sample set, the counts per minute is plotted against the amount of mitochondrial DNA added. The plot is linear and the negative average of the values for the three intercepts on the x-axis yields the amount of nanograms of mitochondrial DNA per microgram of total cell DNA. The method is highly reproducible with a standard deviation of approximately 9 percent. The advantages of using this method over others that have been reported are discussed.

Animals↗
PubMed1988

Cytoplasmic suppression of tumorigenicity in reconstructed mouse cells.

Previous cybrid studies aimed at demonstrating cytoplasmic suppression of tumorigenicity have been generally inconclusive because of (a) the use of mutagens or carcinogens to introduce nuclear-coded and cytoplasmic-coded genetic markers and (b) dilution of putative cytoplasmic suppressors with tumorigenic cytoplasm of whole cells used in the cybrid construction. We have circumvented these potential problems by examining tumorigenicity in reconstructed cells made from tumorigenic karyoplasts and nontumorigenic cytoplasts and by using a ricin-antiricin selection to obtain the reconstructed cells. Karyoplasts from tumorigenic NIH/3T3 cells that were derived from a clone that had survived incubation with benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxy (anti) and been passaged 17 times were fused to NIH/3T3 cytoplasts derived from nontumorigenic cells. The cytoplasts were loaded with antiricin antibody prior to fusion. Ten clones which survived ricin selection were not tumorigenic in nude mice. These findings offer support for the presence of cytoplasmic factors in nontumorigenic mouse cells that suppress benzo(a)pyrene epoxide-induced tumorigenicity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Cytoplasmic suppression of tumor progression in reconstituted cells.

This report details studies of whether mouse NIH/3T3 TGr karyoplasts that are exposed to benzo[a]pyrene epoxide(trans) (BPDE) can progress to tumorigenicity when they are rescued with either mouse B10mtJ CAPr tumorigenic (experiment 1) or nontumorigenic (experiment 2) cytoplasts. The mitochondrial DNA of the B10mtJ cells has restriction fragment length differences that allow distinction from the mitochondrial DNA of the NIH/3T3 cells. The reconstructed clones in experiment 1 were all tumorigenic, while those from experiment 2 were all nontumorigenic. The clones in both experiments were passaged for an equivalent time. These findings reflect the presence of factors in mouse cytoplasm capable of suppressing the tumor phenotype of NIH/3T3-BPDE treated karyoplasts when rescued at an early stage of progression.

Animals↗
PubMed1986

Effects of normal human fibroblast mitochondrial DNA on segregation of HeLaTG Mitochondrial DNA and on tumorigenicity of HeLaTG cells.

We isolated hybrids and cybrids using HeLaTG cells and human normal primary fibroblasts to examine the functional differences between the mitochondrial genomes of tumor and normal cells with respect to their possible involvement in the regulation of tumorigenicity. Hybrids contained mitochondrial DNA (mtDNA) predominantly from the fibroblast parent and their tumorigenicity was suppressed completely. Then, cytoplasmic transmission of primary fibroblast mtDNA to HeLaTG cells was carried out using toxin-antitoxin selection. Two cybrid clones containing a HeLaTG nucleus only and more than 60% of transmitted fibroblast mtDNA were isolated and injected into nude mice to test their tumorigenicity. They formed tumors when 2 X 10(6) cells were injected, whereas no tumors were formed after injection of 5 X 10(5) cells (a concentration at which HeLaTG subclones formed tumors). These cybrids were cultivated in normal medium for two additional months and the content of fibroblast mtDNA increased gradually, resulting in HeLaTG mtDNA eventually being lost from both cybrid clones. We again examined their tumorigenicity and found that they recovered tumorigenicity completely. These results indicate that tumorigenicity of HeLaTG cells could not be suppressed by replacing their mitochondrial genomes with those of normal primary fibroblasts. Further, the partial suppression of tumorigenicity observed in the cybrid clones was temporary and may be due to cytoplasmic factors other than the mitochondrial genomes. Although we can find no difference between the mitochondrial genomes of normal and tumor cells regarding the regulation of tumorigenicity, the segregation pattern of the mtDNA in the cybrids was of interest: in the absence of any mitochondrial selection, HeLaTG mtDNA was lost while fibroblast mtDNA was retained, even though the nuclear component of these cybrids was from the HeLaTG cells. Thus, there should be some functional differences between the mitochondrial genomes of HeLaTG cells and primary fibroblasts that are responsible for the preferential segregation of HeLaTG mtDNA from the cybrids.

Animals↗
PubMed1985

Hydroxyl radicals do not crosslink a DNA-lysozyme complex.

The ionic complex between lysozyme and either Escherichia coli DNA or pBR322 DNA was not crosslinked by two systems capable of producing nanomolar amounts of hydroxyl radicals, the oxidation of xanthine by xanthine oxidase and the iron catalyzed oxidation of ascorbic acid. Nor did effective crosslinking occur with micromolar quantities of hydroxyl radicals raised by the addition of adenosine nucleotides to ferrous iron and hydrogen peroxide. In this case, radical content was estimated by colorimetric analysis of formaldehyde following hydroxyl radical oxidation of dimethyl sulfoxide. Similar amounts of radicals generated by pulse radiolysis in a nitrous oxide atmosphere failed also to induce crosslinking. These findings do not support a role for hydroxy radicals in the N-acetoxy-2-acetylaminofluorene induced crosslinking of DNA to lysozyme proposed earlier.

Cross-Linking Reagents↗
PubMed1983

Evidence that N-acetoxy-N-acetyl-2-aminofluorene crosslinks DNA to protein by a free radical mechanism.

A method was devised to quantitate N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) induced crosslinking of the ionic complex between [methyl-3H]thymidine labeled pBR322 DNA and lysozyme. This involved chromatography of the modified complex on Sephadex CM 25 with a high salt buffer that dissociated the non-covalently bound complex and permitted early elution of unbound [3H]DNA. The crosslinked complex was then eluted with buffer at pH 10.5. The amount of crosslinking was a function of the carcinogen concentration in the reaction mixture containing the complex. Addition of the spin trap nitrosobenzene or the radical trap 2,2-diphenyl-1-picrylhydrazyl to the complex prior to the addition of N-AcO-AAF decreased crosslinking. Reaction of the carcinogen with a solution of [methyl-3H]thymidine labeled Escherichia coli DNA led to the formation of tritiated water that was azeotropically distilled from the mixture. If nitrosobenzene was added prior to the carcinogen, the amount of tritium water released was depressed. Addition of N-AcO-AAF to a mixture of acrylamide and bisacrylamide increased its relative viscosity. Far u.v. irradiation of the complex or reacting it with ammonium persulfate that dissociates to the sulfate anion radical, SO(4), induced crosslinking. While these observations support a free radical mechanism for crosslinking, whether the free radicals arise from heterolytic or homolytic cleavage of N-AcO-AAF remains undetermined.

2-Acetylaminofluorene↗
PubMed1980

Evidence that deoxyribonucleic acid photolyase from baker's yeast is a flavoprotein.

DNA photolyase purified from baker's yeast by affinity chromatography on UV-irradiated DNA noncovalently bound to cellulose and by chromatography on activated thiol-Sepharose 4B yields a single protein band having a molecular weight of 51 000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight, 53 000, determined by gel filtration was in good agreement. Upon denaturation of photolyase by heat or 8 M urea, flavin adenine dinucleotide (oxidized) was isolated from the mixture and identified by thin-layer chromatography and spectral analysis. In contrast to flavoproteins to which flavin adenine dinucleotide (oxidized) is bound which generally exhibit two absorbance maxima between 300 and 500 nm, photolyase has only one at 380 nm. These findings and the similar characteristics of the absorbance and emission spectra of native photolyase with those of flavoproteins in which the chromophore is considered to be the 4a,5-reduced flavin have led us to propose this configuration for the photolyase chromophore. The difference in properties of yeast photolyase compared to the one reported previously supports the idea that there are two photolyases in baker's yeast.

Deoxyribodipyrimidine Photo-Lyase↗