Deuterium oxide (heavy water) arrests the cell cycle of PtK2 cells during interphase.

Deuterium oxide (D2O, heavy water) exerts an antiproliferative effect on a variety of cells in vitro and on some organisms. This effect is mainly ascribed to a tubulin-mediated antimitotic action. We evaluated the morphology, the mitotic activity, and the dynamics of the cell cycle of PtK2 cells grown in vitro in the presence of 75% D2O for up to eight weeks by microspectrophotometric DNA measurements as well as flow cytometric analysis and a determination of mitotic indices. Substitution of heavy water for water in the culture medium initially increased the mitotic index by a (pro-) metaphase block but after 2 to 3 days of incubation no mitotic figures were seen. Analysis of cells grown for 6 days in medium containing 75% D2O revealed accumulation of cells in S/G2-phase. Extended treatment stabilized the high level of cells in this specific phase, when compared to normal growing cells. Cells grown for 1 to 6 weeks in the presence of D2O remained non-proliferating, nevertheless, they were able to divide again after recovery in non-deuterated medium. The time needed for resumption of the mitotic activity was proportional to the duration of deuterium oxide exposure. Cells incubated for 8 weeks in 75% D2O did not recommence mitotic activity. Light and electron microscopic examination revealed characteristic morphological changes of size and ciliation in PtK2 cells subjected to prolonged deuteration.(ABSTRACT TRUNCATED AT 250 WORDS)

Extinction of the HPV18 upstream regulatory region in cervical carcinoma cells after fusion with non-tumorigenic human keratinocytes under non-selective conditions.

'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non-tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non-malignant human keratinocytes are responsible for the extinction phenomenon.

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

Comparison of S-phase fractions measured by flow cytometry and autoradiography in human transplant tumors.

The 3H-thymidine labeling index (TLI) and the percentage of cells in the S-phase have been determined by autoradiography and by flow cytometry, (FCM), respectively, in six malignant tumors of human origin transplanted on athymic nude mice. The Dean and Jett model and the graphical model were used to determine the percent of S-phase cells by FCM. Cell cycle analysis was performed using 1) no correction for background; 2) an algebraic function for background correction; and 3) an exponential function for background subtraction. Each of these three data sets was evaluated using both the Dean and Jett model and a graphical model for the evaluation of DNA histograms. The S-phase fractions (SPF) were compared to the corresponding labeling index results. SPF without background correction were 1.54 times higher than the TLI. SPF, after correction using the algebraic model, were 1.29-fold higher than the TLI, whereas SPF obtained after background subtraction according to the exponential model were only 1.05-fold higher than the TLI. Student's t-test revealed significant differences between the mean TLI values (16.25 +/- 9.06) and the mean SPF obtained by FCM without background correction (mean 25.0 +/- 9.36, P less than 0.01), but not between the mean TLI values and the mean SPF percentages after algebraic (mean 21.0 +/- 10.29) and exponential background correction (mean 17.11 +/- 11.59), P greater than 0.05 each. There was no difference between the results obtained using the Dean and Jett model and those obtained using the graphic evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytostatic and cytotoxic response of Ehrlich ascites tumor cells in vivo on chronic treatment with cytarabine, bleomycin, and peplomycin.

The cytokinetic response of Ehrlich ascites tumor (EAT) cells in vivo upon chronic treatment at low dosage levels with cytarabine (1-beta-D-arabinofuranosylcytosine, ara-c) bleomycin (BLM) and peplomycin (PEP) was estimated. Bivariate DNA histograms allow the simultaneous evaluation of the cell cycle status of living and killed cells. It could be confirmed that ara-C is cytostatic on cells in S phase. Pronounced cytotoxicity was observed in G1 and G2+M phase. BLM and PEP showed no (or neglectable ) accumulation of vital cells in any cycle phase. Both drugs, however, are cytotoxic on cells, regardless their position within the cell cycle. A successive application of ara-C and BLM (or PEP) in a cell kinetics-directed therapy schedule may be taken into account.

A flow cytometric study of chromosomes from rat kangaroo and Chinese hamster cells.

Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was otimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated inot 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in teh karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing hihg power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influences by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.

Visualization of multidimensional spectra in flow cytometry.

Flow data from a cell sorter have been processed by hardwired circuits which include amplification, discrimination, coincidence requirements, peak sensing and holding, A-D conversion, and a computerized pulse height analysis with storage of the spectra obtained. Two dimensional spectra can be stored directly in memory, on tape and disk. Three and four parametric cellular events can be recorded on line during the flow measurement in a sequential mode on tape for subsequent recall. Simple processing of these data can be performed for displaying of two dimensional projections from these multidimensional spaces based on threshold conditions for the remaining parameters. Interfaced transmission of the stored data to a large scale computer enables more sophisticated data analysis. Data reduction by means of a multidimensional probability analysis has been carried out in order to transfer the spectra to a computerized picture system for display. This system creates perspective two-dimensional images from a three-dimensional data space. Frequency can be converted into grey levels. Hard copy in color (color as the third dimension and color intensity as frequency) simplifies the visualization of multiparametric flow data sets.

Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.

Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combination revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest. With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.