Determinants of DNA yield and quality from buccal cell samples collected with mouthwash.

Buccal cells are becoming an important source of genomic DNA in epidemiological studies, but little is known about the effect of different sampling conditions on DNA quality and yield. We used a mouthwash protocol to collect six daily buccal cell samples from 35 healthy volunteers. Twenty-four individuals (six men and 18 women) correctly completed the protocol and were included in paired analyses to determine whether "swish" time (30 s versus 60 s), toothbrushing before collection, or lag time between collection and DNA extraction (1 day versus 5, 10, or 30 days at room temperature) would affect sample quality and yield. Total DNA, human-specific DNA (hDNA), degradation of DNA, and ability to amplify by PCR were determined. hDNA yield did not significantly vary by "swish" time. However, toothbrushing 1 h before sample collection reduced the amount of hDNA by nearly 40% (34 microg versus 21 microg; P = 0.06). Median hDNA yields for samples that were held for 1, 5, 10, and 30 days before extraction were 32 microg (range, 4-196), 32 microg (2-194), 23 microg (3-80), and 21 microg (5-56), respectively. The 10- and 30-day samples had significantly less hDNA than those processed after 1 day (P = 0.01). PCR success rates for beta-globin gene fragments of length 268 bp, 536 bp, and 989 bp were 94% or better, and high molecular weight DNA (>23 kb) was found in all but one sample. These results suggest that buccal cells should be collected before brushing teeth and processed within 5 days of collection to maximize hDNA yield.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Collection of buccal cell DNA using treated cards.

We devised a simple, noninvasive, cost-efficient technique for collecting buccal cell DNA for molecular epidemiology studies. Subjects (n = 52) brushed their oral mucosa and expectorated the fluid in their mouths, which was applied to "Guthrie" cards pretreated to retard bacterial growth and inhibit nuclease activity (IsoCode, Schleicher and Schuell, Keene, NH). The cards are well-suited for transport and storage because they dry quickly, need no processing, and are compact and lightweight. We stored the samples at room temperature for 5 days to mimic a field situation and then divided them into portions from which DNA was extracted either immediately or after storage for 9 months at room temperature, -20 degrees C, or -70 degrees C. The fresh samples had a median yield of 2.3 microg of human DNA (range, 0.2-53.8 microg), which was adequate for at least 550 PCR reactions. More than 90% of the samples were amplified in all three beta-globin gene fragment assays attempted. DNA extract frozen for 1 week at -20 degrees C also performed well. Stored samples had reduced DNA yields, which achieved statistical significance for room temperature and -70 degrees C, but not -20 degrees C, storage. However, because all of the stored samples tested were successfully amplified, the observed reduction may represent tighter DNA fixation to the card over time rather than loss of genetic material. We conclude that treated cards are an alternative to brushes/swabs and mouth rinses for the collection of buccal cell DNA and offer some advantages over these methods, particularly for large-scale or large-scale or long-term studies involving stored samples and studies in which samples are collected off-site and transported. Future studies that enable direct comparisons of the various buccal cell collection methods are needed.

Identity of original and late passage Dami megakaryocytes with HEL erythroleukemia cells shown by combined cytogenetics and DNA fingerprinting.

During routine authentication checks, we noticed untoward karyotypic similarities between late-passage stocks of the Dami megakaryocyte and HEL erythroleukemia cell lines. Genetic identity of Dami with HEL was demonstrated by DNA fingerprinting with a (gtg)5 multilocus probe and confirmed for earlier passages of Dami deposited by its originators with the ATCC. As initial passage stocks of Dami are no longer available for comparison, we investigated whether cross-contamination by HEL was more likely to have occurred during Dami's establishment or subsequently, by karyotyping currently available stocks of both cell lines for comparison with that originally reported for Dami. We found that the karyotype of current stocks of Dami overwhelmingly resembles that described in the original report, having retained at least 16/18 structural chromosome rearrangements as described therein, cf. 12/20 shared by current stocks of Dami and HEL. HEL's antecedence is shown by the retention of normal homologs of chromosomes, 6, 18, and 21--all rearranged in Dami. These results confirm the identity of current and early passage stocks of Dami and their common origin by cross-contamination with HEL which had occurred by July 1987, a year prior to publication. Thus, most, if not all, studies using Dami are likely to have employed HEL instead and may require reappraisal.

Cell line banks and their role in cancer research.

The utility of centralized cell banks in providing reference cultures for cancer research is reviewed. Procedures applied at The American Type Culture Collection in development, maintenance and expansion of such a resource are discussed for example, with emphasis on human tumor cell lines. The various categories of cell-line holdings are explained, and status with regard both to the numbers of lines available and distribution experienced are documented. The locations of other national cell repositories plus contact data are provided.

Lipopolysaccharide-induced differential cell surface expression of intercellular adhesion molecule-1 in cultured human umbilical cord vein endothelial cells.

The effect of endotoxin on cell surface ICAM-1 expression in human umbilical cord vein endothelial cells (HUVEC) was examined using solid phase radioimmunoassay, immunocytochemistry and electron microscopy. At various incubation times (e.g. 3, 6, 12, 24 h), the ICAM-1 expression was enhanced by lipopolysaccharide (LPS, or endotoxin) from one ng/ml to 100 micrograms/mL with maximal enhancement at .1-1 micrograms/mL. The kinetics at 1 microgram/mL LPS showed that the maximum ICAM-1 expression occurred at 24 h. The LPS-induced ICAM-1 expression was not inhibited by the neutralizing rabbit polyclonal antibodies against human IL-1 alpha, IL-1 beta, and TNF alpha, either alone or in combination. This indicated that the mechanism of this induced expression was not an autocrine effect mediated by the LPS-induced IL-1 or TNF alpha. The LPS-induced cell surface ICAM-1 exhibited a polarized distribution shown in immunoelectron micrographs with higher density on the luminal surface. DNA synthesis activity of HUVEC was, contrary to the ICAM-1 expression, suppressed by LPS. Immunocytochemical studies indicated that ICAM-1 was not uniformly expressed in the culture, i.e., some cells expressed more surface ICAM-1 than others, and some of the ICAM-1-expressing cells had an uneven patchy distribution of this antigen. Combined immunocytochemical and [3H]thymidine incorporation studies showed that cells with strong ICAM-1 expression had little DNA synthesis activity, while those with little ICAM-1 expression synthesized DNA actively. ICAM-1 on endothelial cells serves as an anchor for the leukocytes in cell-cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of DNA fingerprints for cell-line individualization.

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

Isolation and characterization of a monoclonal antibody specific for epithelial cells.

Monoclonal antibodies were generated to antigens on human foreskin keratinocytes to identify epithelial-specific molecules. Spleen cells from BALB/c mice, immunized with membrane preparations from primary explants of foreskin epithelial cells, were fused with the NS-1 mouse myeloma line. Hybridoma supernatants were screened for the desired immunological reactivity using enzyme-linked immunosorbant binding assays. Hybridomas secreting antibodies reacting with epithelial cells, but not fibroblasts or lymphocytes, were cloned by limiting dilution, and two stable clones producing immunoglobulin M K antibodies were selected for study. Evaluation of fixed paraffin-embedded human tissue by an indirect immunoperoxidase technique revealed that the antibodies bound most strongly to normal stratified squamous and transitional epithelium, and squamous and transitional cell carcinomas. Antibodies from the cloned hybridomas also reacted with primary cell cultures of foreskin keratinocytes, pulmonary epithelium, fetal liver, and amnion cells, but not with primary cultures of nonepithelial cells. Further testing by enzyme-linked immunosorbent assays revealed that the antibodies reacted with some long-term cell lines derived from epithelial tumors. Nonepithelial cell lines were not stained by the antibodies. Indirect immunofluorescent studies indicated that staining was confined to the cell surface. These antibodies may prove useful in studies of differentiation markers of human epithelial cells.

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum.

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.

Isolation and characterization of monoclonal antibodies reactive with endothelial cells.

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.