Biomedical subjects

D B Krizman

Publications and source records attributed to D B Krizman.

34 records · Page 2Linked to original sources

PubMed1995

Identification of 3'-terminal exons from yeast artificial chromosomes.

We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.

Animals↗

PubMed1993

Efficient selection of 3'-terminal exons from vertebrate DNA.

Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3'-terminal exons. A copy of the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600-700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.

Animals↗

PubMed1992

Correlation of increased levels of Ha-ras T24 protein with extent of loss of gap junction function in rat liver epithelial cells.

Although it is known that cells transformed by ras and other oncogenes show reduced gap junction function, to date there has been no investigation of the quantitative relationship between intracellular levels of ras oncoprotein and loss of cell-cell communication. Using the rat liver epithelial cell line MTR6, which carries a zinc-inducible metallothionein ras T24 (MTrasT24) fusion gene, we showed a direct correlation between the accumulation of ras T24 protein and the loss of dye transfer as measured by interactive laser cytometry. After stimulation with zinc sulfate, changes in both parameters were rapid and measurable by 24 h. Similarly, there was a dose-response relationship between loss of gap junction function and increase in ras T24 protein. Northern analysis of two gap junction proteins (connexins 43 and 32) showed no differences between cells that expressed high levels of ras and control cells. These data demonstrate that the degree of loss of gap junction function is dependent on the amount of increase in ras T24 protein levels, but the mechanism by which these changes are effected remains unclear.

Animals↗

PubMed1992

Linkage map of human chromosome 9 microsatellite polymorphisms.

Ten microsatellite markers composed of polymorphic (CA)n or (AAAT)n repeats were mapped to chromosome 9. PIC values for these markers ranged from 0.46 to 0.82. The marker at the D9S54 locus was localized to 9pter-p22 by means of a somatic cell hybrid; another marker at D9S103 was similarly localized to 9q34-qter. Two-point lod scores and individual meiotic recombination events were used to position the 10 markers relative to each other. The best order resulting from these analyses was D9S54-D9S104-[D9S52-D9S43-D9S50]-D9S53+ ++- [D9S106-D9S105]-D9S51-D9S103, with order of the loci within brackets uncertain. Two-point linkage analysis was also used to approximate the positions of the microsatellite markers relative to those of 33 markers contained in the public CEPH database (v.3) and to one other available microsatellite marker at the D9S15 locus.

Alleles↗

PubMed1991

9p monosomy in a patient with Gilles de la Tourette's syndrome.

Gilles de la Tourette's syndrome (GTS) is a genetic disorder characterized by multiple motor and vocal tics, obsessive-compulsive disorder, and attention-deficit disorder. Family studies support the presence of an autosomal dominant gene; however, to date, an assignment for the GTS locus has not been made. We present the case of a boy with GTS and a deletion of the terminal portion of the short arm of chromosome 9, del(9)(qter----p2304:).

Adolescent↗

PubMed1991

Susceptibility to ras oncogene transformation is coregulated with signal transduction through growth factor receptors.

The human teratocarcinoma cell line PA-1 was derived from culturing ascites fluid cells from a patient with an ovarian germ line tumor. We previously described a non-neoplastic variant cloned from the PA-1 human teratocarcinoma cell line, clone 6, which at passage 40 was resistant to transformation by activated ras oncogenes. However, these cells could be transformed by a plasmid containing both myc and ras. Another PA-1 cell variant, clone 1, isolated at passage 63 and used 50 passages later becomes tumorigenic in nude mice after transfection with an activated ras oncogene (Tainsky et al., Anticancer Res., 8, 899-914, 1988). We report here that the progression from ras resistance to ras susceptibility occurs in both clone 1 and clone 6 cells during 25 passages in culture. In the presence of epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor, the ras-transformable cells exhibit anchorage independent growth, whereas the ras-resistant cells can not be growth stimulated by these growth factors. Similarly, ornithine decarboxylase (ODC) activity was inducible in ras susceptible and ras transformed cells by these growth factors, but not in the ras resistant cells. These differences are not due to the level and activity of epidermal growth factor receptor or to the level of expression of 25 proto-oncogenes.

Blotting, Northern↗

PubMed1991

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.

Blotting, Northern↗

PubMed1990

Changes in rasT24 expression do not induce changes in c-jun, jun-B, or jun-D RNA levels in rat liver epithelial cells.

We used a series of rat liver epithelial (RLE) cell lines that carry a zinc-regulatable metallothionein/rasT24 fusion gene (MTrasT24) to investigate the relation of ras oncogene expression to steady-state RNA levels of the jun family of genes. In these cells, steady-state RNA levels of c-jun, jun-B, and jun-D were unrelated to rasT24 RNA levels or the phenotypic changes induced by the ras oncogene. Steady-state levels of the three jun mRNAs varied among different rasT24 transformed clones, and, although some clones exhibited concomitant induction of rasT24 and jun mRNAs, other clones exhibited no such correlation. We conclude that the effects of rasT24 in transformed RLE cells do not appear to be mediated by c-jun, jun-B, or jun-D and that studies examining only a single transformed clone may give misleading results with respect to the role of various oncogenes in the transformation process.

Animals↗

PubMed1990

Susceptibility for N-ras-mediated transformation requires loss of tumor suppressor activity.

We have been using PA-1 human teratocarcinoma cells to study mechanisms by which oncogenes induce transformation. Tumorigenic PA-1 cells at passages greater than 100 (greater than P100) contain a spontaneously activated N-ras oncogene, while earlier-passage preneoplastic cells contain only the germ-line protooncogene and are nontumorigenic. One preneoplastic cell clone of PA-1 cells can be transformed by introduction of the cloned PA-1 N-ras in gene-transfer experiments, while another earlier-passage clonal cell line cannot be transformed. The goal of this investigation was to determine how human cells progress from resistance to susceptibility to ras oncogene-induced transformation. Somatic cell hybridization experiments described in this report indicate that the resistance of the low-passage cells to transformation is a dominant trait suppressing transformation. Loss of chromosomes from hybrid segregants suggested that tumor suppressors exist on chromosomes 1, 4, and 11. Extended in vitro passaging of somatic cell hybrids also resulted in the loss of chromosomes. Chromosome 1 was lost in these populations of cells, implying that reduction of this chromosome may promote proliferation and not specifically affect tumor formation.

Blotting, Southern↗

PubMed1987

Genetic instability in fibroblasts of patients with thyroid cancers (TC).

In cultured fibroblasts initiated from fresh tumor biopsies of 11 patients with thyroid tumors, 5 medullary carcinoma of the thyroid (MCT) and 6 papillary follicular, the chromosomes are relatively unstable when compared with those from control fibroblast cultures. Tumor-derived fibroblast samples showed a mean number of cells in metaphase with chromosome abnormalities at a frequency of 9.1% and a range of 6-13%, whereas 5 controls exhibited a mean frequency of 4% and a range of 3-5%. Our results indicate that genetic instability in thyroid cancer patients is present not only in peripheral blood cultures, as reported earlier, but in their fibroblast cultures as well.

Cells, Cultured↗

PubMed1985

Double minutes in the HeLa cell line.

Metaphase preparations of three sublines of the HeLa line showed the presence of double minutes (DM) in varying frequencies. In two sublines (S3 and TCH-3753), the size of the DM was variable, whereas in the Fe-1000 subline, they were uniform. Giemsa banding preparations revealed typical HeLa marker chromosomes in all sublines.

Cell Line↗

PubMedDate not supplied

Multiple phenotypic divergence of mammary adenocarcinoma cell clones. I. In vitro and in vivo properties.

The properties of cell clones derived from locally growing and spontaneous metastases of 13762NF mammary adenocarcinoma change during in vitro growth. This has been termed phenotypic drift and is reproducible in independent experiments using different cryoprotected cell stocks. To determine whether phenotypic drift in 13762NF cell clones is the result of an en bloc shift in the properties of all tumor cells, or independent phenotypic divergence of tumor cells to produce a mixed cell population, local tumor-derived clone MTF7 was subcloned at low and high culture passage numbers in vitro. Each subclone was analyzed in vitro for cell morphology, growth rate, saturation density, karyotype and ploidy, and in vivo for experimental metastatic behavior. Subclones derived from low passage clone MTF7 (T11; tissue culture passage number 11) were relatively homogeneous in their growth rates (doubling times of 16.8-17.4 h) and saturation densities (approximately 2 X 10(5) cells/cm2); yet, these same subclones were heterogeneous in their in vitro cell morphologies, experimental metastatic potentials (means range from 0 to greater than 100 tumor nodules per lung), size distributions of lung tumor nodules, marker chromosomes and modal chromosome numbers. High passage MTF7 (T35; tissue culture passage number 35) subclones had similar growth rates and saturation densities, except for subclone 2, which had a doubling time of approximately 26 h. Cell morphologies, experimental metastatic potentials (means range from 3 to greater than 600 tumor nodules per lung), size distribution of lung tumor nodules, marker chromosomes and modal chromosome numbers varied between MTF7 (T35) subclones. The results suggest that simultaneous, independent divergence of several phenotypes from a single cloned cell occurred to form a mixed cell population containing cells with independently segregated, unrelated phenotypes. Thus, the reproducibility of phenotypic drift in clonal cell populations was probably the result of tumor cell divergence and was not an en bloc shift in phenotypic properties of all cells.

Adenocarcinoma↗

PubMedDate not supplied

Picoliter-scale protein microarrays by laser direct write.

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.

Equipment Design↗

PubMedDate not supplied

The role of tissue microdissection in cancer research.

Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.

DNA, Neoplasm↗

PubMedDate not supplied

Quantitative study of a proposed interstitial del (20p 12.2) in multiple endocrine neoplasia (MEN-II).

A quantitative measurement of the prophase Giemsa banding patterns in chromosome 20 of the peripheral blood samples obtained from seven patients with medullary carcinoma of the thyroid (MCT), three nonsymptomatic family members of MCT patients, and three normal controls has revealed no differences in the short arms of the two homologs. More specifically, measurements of band from 20p 12.1 to 20p 12.3 and statistical analysis of the mean ratio between these bands have given no indication of intersitial deletion in chromosome 20p of MCT patients.

Carcinoma↗

PubMedDate not supplied

PA-1, a human cell model for multistage carcinogenesis: oncogenes and other factors.

We have developed a cell system which utilizes the human teratocarcinoma cell line PA-1, from which we have characterized four stages of tumor progression. Soon after establishment in culture PA-1 cells revert and are no longer tumorigenic in athymic nude mice. Later, PA-1 cells as they are passaged in culture, become tumorigenic at passage 100. The transition from nontumorigenic to tumorigenic is the result of the biological effects of an activated N-ras oncogene and can be reproduced by transfection of the cloned oncogene into preneoplastic PA-1 cells. Certain preneoplastic cells (prior to passage 100) in this series are susceptible to transformation by single oncogenes while others are not. In studying the basis of this susceptibility to single oncogene induced transformation we have found that somatic cell hybrids between preneoplastic cells which can suppress ras-induced transformation and ras-transformed cells are non-tumorigenic. Therefore, we believe that the progression from ras suppressing to ras susceptibility may be due to the inactivation of a trans-dominant suppressor gene. Our system has identified at least three steps which lead to tumorigenicity; establishment of growth past senesence, activation of a ras oncogene, and inactivation of an oncogene suppressor function. Further genetic alterations are necessary for tumor dissemination and metastasis.

Cell Line↗