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L S Cram

Publications and source records attributed to L S Cram.

At least 37 records · Page 2Linked to original sources
PubMed1989

The clinical usefulness of chromosome analysis by flow cytometry.

Historical and clinical aspects of chromosome analysis by flow cytometric methods are reviewed. A new method of preparing small samples (1.0 mL of blood) of peripheral lymphocytes for flow karyotype analysis using phytohemagglutinin and interleukin-2 is presented in detail. Figures of flow karyotypes, partial banded karyotypes, and idiograms of patients with inv(8), rec dup(8), rob t(14;21), and t(1;22) are presented, as well as examples of univariate and bivariate flow histograms from other researchers' published works. The clinical utility of these techniques is explored, with specific reference to recent work in chromosome polymorphisms and cultured amniocyte lines. We conclude that, although flow karyotyping is not a replacement for classical banded chromosome analysis, used as an adjunct, this new technique has some clinical usefulness relating to its capability to resolve small differences in chromosomal DNA content.

Animals↗
PubMed1988

Spontaneous in vitro neoplastic evolution of cultured Chinese hamster cells. Nucleolus organizing region activity.

Silver staining to demonstrate active nucleolus organizing regions (NORs) was performed at four different stages of the spontaneous tumorigenic progression in vitro of Chinese hamster WCHE/5 cells. The number of active NORs increased for fully transformed, highly tumorigenic, late passage cells. The increase of NOR material was due to additional NOR-bearing chromosomes or chromosome arms, i.e., trisomy 5, trisomy 8, and the marker chromosome i(3q). Intermediate stages of the neoplastic evolution showed changing patterns of NOR activity, but not an overall increase. We postulate that the increase of active rDNA enhances cell growth and provides undefined selective advantage, and that this supports our previous conclusion that selectable karyotype changes provide competitive advantages rather than being essential for neoplastic evolution in vitro.

Animals↗
PubMed1988

A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.

A highly conserved repetitive DNA sequence, (TTAGGG)n, has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.

Base Sequence↗
PubMed1988

Characterization of human heterochromatin by in situ hybridization with satellite DNA clones.

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.

Avidin↗
PubMed1987

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis.

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.

Base Sequence↗
PubMed1987

Karyotype instability of Chinese hamster cells during in vivo tumor progression.

The extent of karyotype instability in spontaneously transformed Chinese hamster cells was determined after tumor formation by cytogenetic analysis of karyotype heterogeneity. The degree of karyotype heterogeneity among tumors formed in nude mice correlated with tumor latent period. The karyotypes of tumors formed after a short latent period by cells of high tumorigenic potential were similar to each other and to the injected cells. The karyotypes of tumors from cells of low tumorigenic potential and long latents periods were diverse, however. No chromosome aberration was common to every tumor. These results suggest that preneoplastic cells whose phenotypes are not directly capable of tumor formation can progress in vivo and that karyotype instability plays an important role in providing cell variants for tumor progression.

Animals↗
PubMed1987

Spontaneous in vitro neoplastic evolution: selection of specific karyotypes in Chinese hamster cells.

Recurrent cytogenetic changes occurred reproducibly in vitro during the spontaneous neoplastic evolution of cultured Chinese hamster cells. In particular, excess 3q material appeared shortly after immortalization in numerous independent trials. By contrast, when clones were isolated at the earliest possible time after immortalization, a wide spectrum of types of cytogenetic evolution followed, which also resulted in transformed and tumorigenic cells. Clones with stable distinct colonial morphologies were used to demonstrate growth rate interactions when subpopulations compete. We conclude that specific recurring karyotypes are associated with specific stem lines with transient growth advantage during the early stages of in vitro carcinogenesis. Stem lines with other karyotypic change or no detectable karyotypic change are almost equally capable of undergoing the entire spontaneous neoplastic process in vitro.

Animals↗
PubMed1987

Cultured Bloom's syndrome substrains: a relationship between growth in low serum and the expression of double minute chromosomes.

Four Bloom's syndrome fibroblast strains were examined for chromosome changes, immortalization, tumorigenicity, anchorage independent growth, and the ability to grow in low serum. Only one strain (GM 1492) exhibited some of these characteristics, which are generally associated with neoplastic cells. Strain GM 1492 also exhibited a low frequency of cells that contained double minute chromosomes. Substrains from GM 1492 were cloned, and showed that some of the above characteristics were expressed as a heritable trait. Karyotype instability (heteroploidy) was seen in all clones studied, with modal chromosome numbers ranging from near-diploid to near-tetraploid, as confirmed by DNA content distributions. Although no clones studied were tumorigenic in nude mice or immortalized, some showed an increased cellular proliferative capacity. Several clones were isolated after growth in 1% fetal bovine serum, and these all showed an increased expression of double minute chromosomes. Two of these 1% serum-isolated clones, (1492/clone 3-1% and clone 8-1%) were further studied by G-banding analysis, and found to show specific chromosomal anomalies. Clone 3 showed monosomy for chromosomes #4, #13, and #19, along with the absence of both copies of chromosome #7 but with both i(7p) and i(7q) chromosomes consistently seen. Clone 8 showed monosomy for chromosome #13. Preliminary experiments using DNA slot blots indicated that erb a and b, v-fes, v-mos, c-myc, n-myc, v-src, and v-sis showed no sequence amplification in GM 1492 cells or subclones.

Animals↗
PubMed1986

Spontaneous in vitro neoplastic evolution: recurrent chromosome changes of newly immortalized Chinese hamster cells.

Spontaneous neoplastic progression in cultured Chinese hamster cells was studied at the earliest stage possible. Eighteen independent newly immortalized cell populations (from six individual Chinese hamsters) were characterized for karyotype instability. Colonies were selected from initial sparse platings of adult or fetal cells and were expanded for study. The chromosomes from these newly established cell lines were studied using a combination of G-banding and flow karyotype analysis. At a slightly later passage, the 18 cell lines were tested for tumorigenicity in nude mice. Frequent recurring chromosome changes were observed in the karyotypes. The most frequent changes were either total or partial trisomy of chromosome #3 (83%) and trisomy of chromosome #5 (61%). Only 4 of 18 clones (22%) were tumorigenic at the time of testing, and these had long latent periods. The presence of recurrent chromosome changes did not obligate these cell lines to become tumorigenic, but the karyotype instability appeared to be an indicator of the ongoing process of neoplasia.

Animals↗
PubMed1986

Spontaneous immortalization rate of cultured Chinese hamster cells.

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.

Animals↗
PubMed1986

Preneoplastic phenotype and chromosome changes of cultured human Bloom syndrome fibroblasts (strain GM 1492).

The Bloom syndrome fibroblast strain, GM 1492, was examined for phenotypic properties generally associated with neoplastic cells. A serial clonogenicity assay indicated that these cells can proliferate in culture, achieving approximately twice the number of population doublings as compared to normal human skin fibroblasts. Strain GM 1492 appeared to be partially transformed in that these cells showed a slight degree of anchorage independence when grown in methylcellulose, and also appeared to have relaxed growth requirements compared to normal fibroblasts. GM 1492 cells are heteroploid, with 20 to 80 chromosomes/cell and a modal chromosome number of 44. Cytogenetic analysis of G-banded metaphase chromosomes indicated that most cells contained at least one copy of each normal human chromosome, and many cells exhibited only aneuploidies with no detectable chromosomal rearrangements. Minute chromosomes were seen in a few of the metaphase cells examined. GM 1492 cells did not form tumors in athymic nude mice. Since many of the characteristics of GM 1492 cells are similar to those seen only in tumor cells, but the strain is nontumorigenic, we suggest that GM 1492 cells are preneoplastic and thus represent an ideal system for the in vitro study of human neoplastic progression.

Animals↗
PubMed1985

Antigen-specific immunostimulation of amino acid transport: proposed method for the diagnosis of tuberculosis in cattle.

Preliminary data on a novel method for the diagnosis of bovine tuberculosis (TB) has been presented. This method is based on increased amino acid transport in specifically stimulated lymphocyte cultures. Lymphocytes isolated from TB-infected or -sensitized cattle and cultured with the TB antigen PPD-B had noticeably enhanced amino acid transport after 1-3 days. Transport continued to increase for up to 5 days of culture with the antigen. PPD-B did not stimulate transport in lymphocytes from nonsensitized animals. The new method correlated well with the tuberculin skin test and antigen-induced lymphocyte proliferation measured by flow cytometry.

Amino Acids↗
PubMed1984

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.

Animals↗
PubMed1984

Flow karyology of serially cultured Chinese hamster cell lineages.

Flow karyology of serially cultured Chinese hamster cell lineages has been observed to be influenced by the degree of cellular heterogeneity in culture. The minimum coefficient of variation (CV) and debris fraction obtainable vary as a cell lineage evolves from a primary cell culture to an established cell line. The cell lineages pass through a stage of decreased cellular heterogeneity from which flow karyotypes can be obtained with lower CV and debris fraction. The influence of cellular heterogeneity on flow karyology is observed with constant preparative protocol and constant instrument performance, and can be an additional source of variability.

Animals↗