Elevated frequencies of benzo(a)pyrene-induced Hprt mutations in internal tissue of XPA-deficient mice.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight and have a high predisposition to developing cancer. At the cellular level, XP patients are defective in nucleotide excision repair (NER). Recently, mice have been generated via gene targeting that are deficient in the expression of the XPA gene [A. de Vries et al., Nature (Lond.), 377: 169-173, 1995]. We have assessed the consequences of defective NER for mutagenesis in normal and XPA mice exposed to benzo(a)pyrene and 2-acetylaminofluorene. To study mutagenesis, mature T lymphocytes were isolated from the spleen and stimulated to proliferate in vitro to select for mutants at the endogenous Hprt locus. Background mutant frequencies in normal and XPA mice were very similar and not influenced by age. Single doses of benzo(a)pyrene administered i.p. resulted in a dose-dependent increase of the Hprt mutant frequency in normal mice. In addition, after chronic exposure to benzo(a)pyrene, Hprt mutants were readily detectable in XPA mice at an early onset of treatment but only at a later stage in normal mice. In contrast, chronic treatment of either normal or XPA mice with 2-acetylaminofluorene did not increase Hprt mutant frequency above the background frequency. This absence of significant induction of Hprt mutants can be entirely attributed to the low frequency of 2-acetylaminofluorene-induced DNA adducts in lymphoid tissue. These results provide the first direct evidence in mammals that deficient NER leads to enhanced mutagenesis in endogenous genes in internal tissue after exposure to relevant environmental mutagens, such as benzo(a)pyrene.

Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.

The role of fibroblastoid cells and macrophages from mouse bone marrow in the in vitro growth promotion of haemopoietic tumour cells.

Adherent cells from mouse bone marrow have been shown to promote the in vitro growth of the AVRij-1 tumour cell line. The experiments presented here suggest that the adherent cells involved in this phenomenon are the progeny of bone marrow derived fibroblastoid colony forming units. The latter were characterized by means of cell density distribution analysis. They had a broad distribution pattern and an average peak cell density of 1.069 g.cm-3. The growth promotion activity exerted by adherent cell layers from the various density fractions on the AVRij-1 tumour cell line coincided with the distribution of fibroblastoid colony forming units. On the other hand, the presence of macrophages in the adherent layers seemed to be non-essential for the in vitro promotion of growth of the AVRij-1 cell line.

Fractionation of normal and beta-thalassemic human hemopoietic progenitor cells by immunomagnetic beads.

Depletion of adherent cells, followed by simultaneous immunomagnetic bead depletion of Leu 4+, Leu 7+, Leu 11+, Leu M1+, Leu M3+, B1+, WEM-G11+, and Glycophorin A+ cells from normal bone marrow mononuclear cells, consistently led to recoveries of erythroid and nonerythroid colony-forming cells of greater than 100% and enrichment of 13- to 99-fold. Similarly, the recovery of mixed erythroid colony-forming cells was greater than 100%, with enrichments of 7.5- to 262-fold. This simple procedure, when used on normal bone marrow and beta-thalassemic peripheral blood mononuclear cells, as well as providing significant enrichment, suggests that colony assays on unfractionated human mononuclear cells specifically underestimate the number of BFU-E, Mix-CFC, and nonerythroid-CFC present.

Characterization of stroma-adherent colony-forming cells: a clonogenic assay for early hemopoietic cells?

Hemopoietic cells that adhered to preformed selected marrow stromal cell layers were characterized on the basis of progenitor cell production capacity, colony-formation kinetics and forward light scatter (FLS) properties. It was shown that early hemopoietic cells attached to the stromal layers within 2 hours of incubation, and were responsible for the initial production of the more differentiated granulocyte-macrophage colony-forming cells (day 14 GM-CFC) in long-term cultures (LTC). In a clonogenic assay system, hemopoietic cells that adhere to stromal layers can be detected by the formation of small colonies of blast-like cells and are designated as stroma-adherent CFC. Cell fractionation on the basis of FLS and counting colonies on days 5, 14 and 21 revealed that there was a succession of colony formation, indicating that the stroma-adherent CFC consisted of subpopulations with different lag-phases before initiation of proliferation. Day 5, day 14 and day 21 stroma-adherent CFC were shown to have a high, intermediate and low FLS, respectively. The cells that produced GM-CFC by day 21 showed FLS properties similar to those of day 21 stroma-adherent CFC, suggesting a correlation between day 21 stroma-adherent CFC and CFC-producing cells in LTC. The CFC present on day 21 required the synergistic action of GM-CSF+IL-3 + stem cell factor (SCF) for optimal proliferation. The prolonged lag-phase, the low FLS and the multifactor-responsive progeny are properties similar to those reported for other early cells, and it is proposed that day 21 stroma-adherent CFC represent an early hemopoietic cell type whereas day 5 stroma-adherent CFC represent a more mature stage of differentiation.

Selection of two human bone marrow stromal cell subpopulations with different effects on the maintenance and differentiation of myeloid progenitor cells.

The simple selection of two human bone marrow stromal cell populations is described. Adherent stromal cell layers were formed in primary cultures of low-density marrow cells. At time of confluence, persistent nonadherent cells were collected and transferred to new culture flasks, where they formed a secondary stromal layer. These primary and secondary stromal layers differed in their ability to support myelopoiesis, as tested by progenitor cell production after inoculation with fresh bone marrow cells. In the presence of primary stromal layers the number of granulocyte-macrophage colony-forming cells (GM-CFC) decreased gradually, but in the presence of secondary layers production of GM-CFC was evident during the first 3 weeks. The regulation of the two stromal types on hemopoietic cell proliferation and differentiation was investigated by determining the kinetics of the transitions within the differentiation sequence of three myeloid progenitor cells. Pre-CFC, day-14 CFC, and day-7 CFC were fractionated by cell sorting on the basis of forward light scatter and cocultured with the two stromal layer types. It was found that the decrease in CFC numbers in the presence of primary stromal layers could be explained by the stimulation of hemopoietic cells into rapid differentiation with loss of proliferative capacity at an early stage of culture. Secondary layers appeared to promote survival and self-renewal of later types of progenitor cells and to trigger more immature cells to proliferate and differentiate at a later time of culture.

The physical separation of three subpopulations of granulocyte/macrophage progenitor cells from mouse bone marrow.

The heterogeneity among progenitor cells of granulocytes and macrophages capable of forming colonies in vitro (CFU-c) is analysed. If specific culture conditions and stimuli are used, it is possible to discriminate among three subpopulations of CFU-c. CFU-c directly responding to CSF prepared from pregnant mouse uteri are characterized by a density of 1.075 g.cm-3. The G1-phase cells of this CFU-c subpopulation have a sedimentation rate of 4.8 mm.h-1. CFU-c of two additional subpopulations are induced to proliferate by enhancing factors present in serum of mice injected with endotoxin 18 hours earlier and in lysate of rat erythrocytes. CFU-c responding to postendotoxin serum have a density of 1.070 g.cm-3 and a sedimentation rate of 4.3 mm.h-1 (G1 cells). The CFU-c responding to erythrocyte lysate have a density of 1.080 g.cm-3 and a sedimentation rate of 5.3 mm.h-1 (G1 cells). The calculated diameters of G1-phase cells of these three CFU-c subpopulations range from 7.4 to 7.6 micron and are not significantly different. It is proposed that the three CFU-c detected represent three sequential maturation stages of granulocyte/macrophage progenitor cells. These cells are similar in size but increase in density (1.070 g.cm-3 to 1.080 g.cm-3) as they mature.

Electrophoretic mobility properties of murine hemopoietic cells in different stages of development.

In vivo spleen colony forming cells (CFUs) and three developmental stages of in vitro granulocyte/macrophage colony forming cells (GM CFUc 1, 2 & 3) are characterized by their surface charge density using the free-flow electrophoresis technique. CFUs have a somewhat higher electrophoretic mobility than have GM CFUc. GM CFUc 1, 2 & 3 appear to be identical in their mobility properties. Treatment with neuraminidase-by which negatively charged N-acetylneuraminic acid (NANA or sialic acid) groups are cleaved from membrane glycoproteins-changes the electrophoretic mobility of the various hemopoietic cells differentially. Neuraminidase treatment strongly reduces the electrophoretic mobility of CFUs and GM CFUc 1, has a moderate effect on the mobility of GM CFUc 2 and causes a relatively small reduction in the mobility of GM CFUc 3. It is suggested that the number of neuraminidase susceptible NANA groups on the cell surface decreases during early hemopoietic cell development.

Effects of neuraminidase treatment on CFUs properties and CFUs numbers.

Incubation of bone marrow cells with Vibrio cholerae neuraminidase decreases the number of CFUs to a minimum of 25% of control numbers. It is shown that this decrease in CFUs numbers is not due to cytotoxic effects of neuraminidase but to a decrease in the spleen seeding efficiency of colony forming cells. Neuraminidase acts in two ways. It removes negatively charged neuraminic acids from membrane glycoproteins and glycolipids and it can adsorb to the cell surface. It is investigated which of the two enzyme effects is responsible for the decrease in CFUs numbers. Inhibition of the enzyme reaction by EDTA prevents to a great extent the decrease in CFUs numbers, while tests with mouse spleen cells and human erythrocytes show that EDTA has no or a positive effect on the adsorption of neuraminidase to the cell surface. This indicates that the neuraminidase induced decrease in CFUs numbers is caused by the enzymatic removal of neuraminic acids rather than by the adsorption of the enzyme to the cell surface.

Characterization and enrichment of murine hemopoietic stem cells by fluorescence activated cell sorting.

Several methods for supravital staining of mouse bone marrow cells are described. These methods were employed to sort fractions of bone marrow cells by a light activated cell sorter and to determine cytochemical properties of hemopoietic stem cells. Labelling with fluoresceinated wheat germ lectin confirmed that the stem cells are among the bone marrow cells with the highest surface density of N-acetylneuraminic acid. Staining with tetracycline revealed that the stem cells contain relatively active or relatively many mitochondria. Staining with the bisbenzimide H33342, a supravital DNA stain, indicated that most pluripotent stem cells are in a G0/G1 state of the cell cycle. Using these and other measurements, separation protocols can be devised to concentrate stem cells. Some of these are discussed.

Cellular cytotoxicity assessed by the 51Cr release assay. Biological interpretation of mathematical parameters.

This study deals with the interpretation of primary data of the 51Cr release assay for cellular cytotoxicity. In particular the dose-effect relationship between increasing numbers of lymphoid cells and the percentage of target cells killed has been considered. The number of target cells killed depends on the frequency and the activity of cytotoxic cells. These two parameters are often not distinguished from each other, which causes confusion and frequently results in vague descriptions of cytotoxicity data. Because in many cases not all cells in the target cell population can be lysed, we recommend the introduction of the plateau value for target cell kill. This maximum of target cell kill is a measure of the frequency of lysable target cells, but also depends on the cytotoxic cell activity. Description of the dose-effect curve by y = A(1 - e-kx) allows the simultaneous calculation of the maximum kill (A) and the frequency of cytotoxic cells (k) in the lymphoid cell population (x). Results are presented which indicate that A and k represent totally independent biological parameters the use of which permits a more objective description of cytotoxicity data.

Rapid expansion of human cytotoxic T cell clones: growth promotion by a heat-labile serum component and by various types of feeder cells.

Culture conditions are described that result in rapid expansion of cloned cytotoxic T cells of human origin. A combination of allogeneic lymphocytes and Epstein-Barr virus (EBV) transformed B cells (B-LCL) as irradiated feeder cells resulted in a 10-fold higher cell yield than obtained by use of either feeder cell alone. The large cell numbers obtained in a relatively short period of time facilitate in vitro and in vivo experimentation. Further enhancement of cell proliferation was obtained by the use of fresh human serum not heat-inactivated before use. This suggests the presence of a heat-labile growth stimulating factor or factors in human serum. Round-bottomed microtitre wells were found to give best culture results. Plating and harvesting of cells cultured in wells was facilitated by a specially designed culture flask. Addition of leucoagglutinin (purified phytohaemagglutinin) to the culture medium resulted in an approximately 3-fold higher cell yield. Optimal culture results were obtained when all the above factors were combined. It was possible to expand single cytotoxic T cells to up to 10(9) cells in about 30 days with full retention of cytolytic activity and target cell specificity. T cell clones have now been cultured for more than 70 generations.

Simple immuno bead labeling of leukemic cell clusters cultured in methylcellulose.

In studies of in vitro leukemic clonogenic cells it is of importance to determine the cell lineage of individual clusters grown in culture. A method is described for the in-situ identification of leukemic cell clusters in methylcellulose cultures. Whole cultures are dried and incubated with various monoclonal antibodies, followed by incubation with beads coated with secondary antibody. Clusters containing antibody-positive cells are heavily labeled with beads which simplifies the recognition and scoring of clusters using normal light microscopy. This method has general applications and can also be used to identify normal myeloid and lymphoid clusters depending on the availability of lineage specific monoclonal antibodies.

Viable bone marrow stromal cells are required for the in vitro survival of B-cell precursor acute lymphoblastic leukemic cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. A number of studies have examined the binding of BCP ALL cells to BM stromal cells and extracellular matrix components. To date there have been no studies examining the effect of such binding on the growth and survival of BCP ALL cells. In this study, by measuring the growth parameters of these cells with use of a lipophilic fluorescent probe, PKH 26 GL, we demonstrate the positive effect of viable BM stromal cells on BCP ALL cell survival in 10 patient samples. At the same time, by comparing these cultures with cultures of the same patient samples in the presence of glutaraldehyde-fixed stromal cells, deoxycholic acid-derived stromal cell matrices, purified laminin, collagen or fibronectin, the role of various stromal cell-derived contact components in BCP ALL survival was tested. It was shown that the survival of BCP ALL cells in vitro was dependent upon viable BM stromal cells present in co-culture as the various contact components did not show any functional effect on BCP ALL cell survival.

Measurement of the growth parameters of precursor B-acute lymphoblastic leukaemic cells in co-culture with bone marrow stromal cells; detection of two cd10 positive populations with different proliferative capacities and survival.

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent probe which becomes incorporated into the plasma membrane upon the staining of cells. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were stained with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cell layers. Viable leukaemic cells from these cultures were identified on the basis of forward light scatter, 90 degrees light scatter, propidium iodide exclusion, PKH 26 GL staining and CD10 expression by flow cytometry at the beginning of the culture period and on days 2, 6, 10 and 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with stromal cells compared with control cultures, without stromal cells. In the presence of stromal cells, survival and proliferation continued throughout the culture period; in contrast in five of seven control cultures no viable cells could be detected after 6-10 days. Interestingly, two populations of leukaemic cells were distinguished on the basis of their different rates of proliferation, when co-cultured with stromal cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stroma-dependent system.

Human bone marrow stromal cell contact and soluble factors have different effects on the survival and proliferation of paediatric B-lineage acute lymphoblastic leukaemic blasts.

Recent studies have confirmed that in vitro viability and proliferation of precursor B-cell leukaemia (ALL) cells are linked to the presence of bone marrow derived stromal cells. To investigate whether this effect is mediated by direct contact or through the action of soluble factors, using a method we have recently described, the growth parameters of ALL bone marrow blast cells from eight newly diagnosed patients were determined with the lipophilic fluorescent probe PKH 26 GL. The viability of ALL cells and the rate of cell division in cultures containing either medium alone; stromal cell conditioned medium; stromal cell layers allowing direct contact, or in 0.4 microns microporous membrane cultures suspended above stromal cell layers were examined. In all eight samples an improved maintenance of ALL cells in a viable state in cultures containing bone marrow stromal cells was observed. The survival of leukaemic cells was equivalent in 0.4 microns microporous membrane cultures suspended above stromal cell layers and in cultures of leukaemic cells in direct contact with stromal cell layers. It was thus demonstrated that this effect was mediated by the action of soluble factor(s) present in these cultures. However, the improved maintenance of ALL cells in a viable state was observed in only one of the eight cases when ALL cells were cultured in stromal cell conditioned medium alone. The highest rate of cell division of leukaemic cells was observed in ALL cells in direct contact with bone marrow stromal cells. The activities of stromal cell derived soluble factors could not be reproduced by recombinant forms of likely candidate factors including IL-1 beta, IL-4, IL-6, IL-7, SCF, TNF alpha, TGF beta, LIF, NGF or a mixture of these factors when examined in cultures of the same patient samples. This study implicates the existence of a novel bone marrow derived factor(s) that improves survival of ALL cells in vitro.

Physical and kinetic properties of haemopoietic progenitor cell populations from mouse marrow detected in five different assay systems.

Properties of haemopoietic progenitor cells detected in several different assays have been compared in order to position them within the haemopoietic developmental lineage. The spleen colony-forming cell (CFUs), the high proliferation potential colony-forming cell (HPP-CFC) and two granulocyte-macrophage colony-forming cells (GM-CFC-1 and GM-CFC-2) have been studied. Two experimental techniques were used: separation of cells on the basis of their buoyant density and comparison of the survival of haemopoietic cells after donor mice had been injected with the cytotoxic drug 5-fluorouracil (5-FU). On linear BSA gradients the modal buoyant densities of CFUs, HPP-CFC and GM-CFC-1 were the same, 1.070 g cm-3; the density of GM-CFC-2 was higher, 1.075 g cm-3. GM-CFC-2 colonies were much smaller and contained far fewer cells than HPP-CFC or GM-CFC-1 colonies, even after prolonged culture, and this suggests that dense haemopoietic progenitors have less proliferation potential. This was confirmed by comparison of the size of colony formed, under identical culture conditions, by progenitors of different densities. Mean colony diameter was inversely related to the density of the progenitor cell. With the exception of GM-CFC-1, low density progenitors were more resistant to the cytotoxic effects of 5-FU than high density precursor cells (GM-CFC-2). Consequently, the GM-CFC-1 could be distinguished from GM-CFC-2 on the basis of buoyant density and from the other low density populations on the basis of post-FU kinetics. The reasons why the GM-CFC-1 should be more sensitive to 5-FU than other low density progenitors are discussed and the relation of these low density precursors to one another in terms of their position within the haemopoietic developmental lineage is elucidated.

A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes.

The application of the fluorescent cell membrane probes PKH2 and PKH 26 GL in the measurement of leukaemic cell growth was examined on four cell lines K562, NALM-6, ACV (a pre-B cell line) and HL-60 using flow cytometry. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. By measuring the mean fluorescence intensity of the cells at the beginning of culture and at various time points, and by combining this information with a viable cell count, it was possible to determine: (1) the number of viable cells; (2) their rate of proliferation; (3) their number of cell divisions; and (4) the maintenance of cells in a viable state over a period of time. It was demonstrated that these parameters could be reliably established using the red fluorescent probe PKH26 GL. In contrast, the green fluorescent probe PKH2 GL showed dye transfer resulting in an underestimation of the number of cell divisions and an overestimation of the maintenance of cells in a viable state. The potential advantages of the use of PKH26 GL over conventional assays for the measurement of leukaemic cell growth parameters are discussed.

Differentiation restriction in the neutrophil-granulocyte, macrophage, eosinophil-granulocyte pathway: analysis by equilibrium density centrifugation.

Bone marrow culture techniques and equilibrium density centrifugation of human bone marrow cells were used to analyse the neutrophil-granulocyte, macrophage and eosinophil-granulocyte progenitor hierarchy. The buoyant density of progenitor cells changes as cells differentiate down the granulocyte-macrophage pathway and this allows the construction of a density 'map' of the points at which differentiation decisions are made. Unipotent progenitors, neutrophil-granulocyte (G), monocyte-macrophage (M), eosinophil-granulocyte (Eo), are more dense than bi- and tripotent progenitors (GM and EoGM) and have a lower 7-day proliferative capacity (assessed as the clone size achieved in maximally stimulated agar cultures). Experiments in which marrow cells were separated on a basis of their density and either cultured in agar immediately or after an interval of 6 days in suspension culture, were performed to establish the density of the cells which give rise to each type of progenitor, i.e. to investigate parent-progeny relationships. In each case the parent cells were of lower density than the unipotent or bipotent progenitor in question. The ability to separate, at least partially, unipotent, bipotent and multipotent cells of closely related lineages is important since it facilitates studies of the intracellular events taking place as restriction of the cell's differentiation options takes place.

Structural abnormalities of chromosome 2 in benign thyroid tumors. Three new cases and review of the literature.

Here we report our cytogenetic findings on three cases of nodular goiter, all showing structural clonal abnormalities of chromosome 2. In the first case, we found a t(2;3)(q21;q27 or q28) in two nodules of the same patient. The second case revealed a t(2;20;3)(p21;q11.2;p25), and the third case showed a t(1;2)(p22;p13). When the data from the literature and the present cases are summarized, the results suggest the existence of at least three breakpoint clusters of chromosome 2 in benign thyroid tumors or hyperplasias.