Viable sorting of intact multicellular spheroids by flow cytometry

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79)

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.     

CS-A therapy in MRL-lpr/lpr mice: amelioration of immunopathology despite autoantibody production

MRL-lpr/lpr mice spontaneously develop massive T cell lymphadenopathy, autoantibodies, and immune-mediated pathology. These mice are thought to be models of various human autoimmune diseases, including systemic lupus, Sjogren's syndrome, and rheumatoid arthritis. We have used cyclosporin A (CS-A) treatment as a tool by which the mechanisms of immune-mediated pathology might be dissected. CS-A was used because of its known preferential inhibition of T cell function and the marked expansion in MRL-lpr/lpr mice of an unusual L3T4-, Lyt-2-, 6B2+ T cell population. CS-A prevented lymphadenopathy and expansion of L3T4-, Lyt-2-, 6B2+ T cells in the peripheral lymph nodes, and also in the thymus. The increased expression of the c-myb and T cell receptor beta-chain genes associated with these unusual cells was also corrected. The finding of increased numbers of L3T4-, Lyt-2-, 6B2+ thymocytes in untreated mice suggests abnormal intrathymic differentiation in lpr/lpr mice, a defect that was corrected by CS-A. Treated mice had a marked decrease in arthritis and glomerulonephritis and significantly prolonged survival. These beneficial effects of CS-A occurred despite a lack of reduction in antibodies reactive with DNA, circulating immune complexes, rheumatoid factor titers, or immunoglobulin concentrations. These results demonstrate that the B cell hyperactivity of MRL-lpr/lpr mice can proceed without the T cell proliferative disease.     

The sesquiterpene lactone hymenoxon acts as a bifunctional alkylating agent

Hymenoxon, a toxic sesquiterpene lactone found in bitterweed, bound deoxyguanosine in a cell free system and formed adducts with guanine residues in cellular DNA. The reactive dialdehyde form of hymenoxon formed stable Schiff base products with deoxyguanosine which were separable from unreacted hymenoxon and deoxynucleosides by reverse phase high pressure liquid chromatography. Hymenoxon adducts which eluted as a single impure peak from the octadecylsilane column separated on amino and diphenyl-bonded phases with 10% methanol. Tritiated nucleoside adducts were isolated and purified from CFW mouse sarcoma cells treated with hymenoxon. Proton nuclear magnetic resonance spectra of purified hymenoxon-deoxyguanosine adducts revealed a loss of signals for hydroxyl groups in the bishemiacetal of hymenoxon. ¹³C-nuclear magnetic resonance spectra revealed that the major adduct has 35 carbon atoms, indicating an interaction of at least two guanine residues per hymenoxon molecule and suggesting that hymenoxon may cross-link DNA. Sedimentation analysis of treated DNA further showed that DNA cross-linking by hymenoxon (30 µg/ml) was equivalent to that of a known cross-linking agent, mitomycin C (7.5 µg/ml). Hymenoxon was more cytotoxic to DNA cross-link repair-deficient Chinese hamster ovary cell mutants than to repair proficient strains. These data combine to indicate that hymenoxon acts as a bifunctional alkylating agent which cross-links DNA in mammalian cells.CHO Chinese hamster ovary - HYM hymenoxon - MMC mitomycin C - NMR nuclear magnetic resonance - PBS phosphate buffered saline     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Cytogenetic studies using Q-band polymorphisms in patients with AML receiving marrow from like-sex donors

Summary After bone marrow transplantation (BMT), it is important to monitor the bone marrow and lymphoid cell populations of the recipient to document engraftment. When donor and recipient are of unlike sex, the sex chromosomes serve as a useful marker to determine cellular origin. When donor and recipient are of like sex, autosomal heteromorphisms can be used to identify the origin of cells in metaphase. Using Q-banding, we found that 17 of 20 patient/donor pairs (85%) examined showed at least one chromosome heteromorphism that distinguished between recipient and donor cells with certainty. Five of the patients were followed up after BMT in order to document engraftment. Donor metaphases could be detected in the marrow within two weeks of BMT when the graft was successful. Chimaerism was detected in the lymphocyte population even when the graft persisted. In a case of graft failure, donor cells did not persist in the marrow, and the lymphocyte population did not convert to donor type. These studies demonstrate that autosomal heteromorphisms are useful in the study of myeloid and lymphoid chimaeric states after BMT.     

Rapid quantitative analysis of a gibberellin-sterol inhibitor using high-performance liquid chromatographic cartridge columns

A rapid, sensitive, and economical chemical analysis of the triazole, gibberellin-inhibitor, paclobutrazol (PP333, [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl) pentan-3-ol]) was sought, featuring high-performance liquid chromatography (HPLC) as the final quantitation step. Three C₁₈-reverse phase columns (conventional, 250×4.6 mm; cartridge type, 125×4.6 mm; and minicolumn, 33×4.6 mm) were evaluated for their performance in HPLC separation and quantitation of PP333 applied to soil and plant foliage. The 125-mm Whatman Partisil 5 ODS-3 cartridge column was superior to the standard 250-mm DuPont Zorbax ODS unit, and provided enhanced resolution and reduced solvent consumption, analysis time, and cost. A Perkin-Elmer Pecosphere 3×3C-C₁₈ cartridge system was also superior to the 125-mm column with respect to these parameters. Although this minicolumn necessitated an additional purification step prior to HPLC analysis, its exceptionally fast analysis time and recovery period coupled with a high degree of sensitivity rendered it the most superior unit. This HPLC technology provided an efficient means of assaying for PP333 in large-scale experiments dealing with the chemical's absorption, translocation, and physiological response.     

Activation of a low specific activity form of DNA polymerase alpha by inositol-1,4-bisphosphate

A low activity form of DNA polymerase alpha immunoaffinity-purified from adult-derived human fibroblasts was activated by interaction with phosphatidylinositol-4-monophosphate, while a high activity form of the enzyme did not interact with phosphatidylinositol-4-monophosphate or its derivatives. Phosphatidylinositol-4-monophosphate was apparently hydrolyzed in the presence of a highly purified low activity form of DNA polymerase alpha, effecting the release of diacylglycerol and the retention of inositol-1,4-bisphosphate by the enzyme complex. The resulting inositol-1,4-bisphosphate/protein complex exhibited increased affinity of binding to DNA template/primer and increased deoxynucleotidyltransferase activity. These data indicate that inositol-1,4-bisphosphate may function as an effector molecule in the activation of a low activity form of human DNA polymerase alpha and suggest that it may function as a second messenger during the initiation of mitosis in stimulated cells.