The effect of region of interest variations on morphologic operations data and gray-level values extracted from digitized dental radiographs.

OBJECTIVE: To determine the correlations among morphologic operations (MO) values and the correlations among gray-level values for regions of interest (ROIs) placed at various locations on digital images of alveolar bone for 45 patients. STUDY DESIGN: As part of a larger study, up to 7 vertical bite-wing radiographs were taken and digitized for each of 45 patients. Sets of 2 rectangular ROIs were placed on the digitized images of interdental alveolar bone at 4 locations for each patient. The ROIs (1 crestal and 1 apical) were placed between second premolars and first molars in all 4 dental quadrants. Gray-level values were measured, and MO analysis was performed on each ROI. Descriptive statistics were calculated and correlations determined. RESULTS: Paired correlations (such as apical vs crestal, left vs right, maxillary apical vs mandibular apical) of MO values were weak (r = 0.01-0.21), but corresponding correlations for gray-level values were relatively strong (r = 0. 60-0.92). CONCLUSION: MO values varied with ROI location considerably more than did gray-level values. Additionally, ROI size and shape apparently affected MO data. Accurate placement and documentation of ROIs appear to be critical considerations in analyses that use MOs.

Retrofitting an esthetic post and core to salvage a six-unit fixed partial denture.

Teeth restored with post and core restorations present a clinical dilemma if they fracture. An individual with a maxillary anterior six-unit fixed partial denture had one of the canine abutment teeth fracture. The other abutment debonded and the post separated from the core. The fixed partial denture was salvaged by retrofitting a porcelain-fused-to-metal post and core restoration in the fractured abutment.

Single K562 human leukemia cells express and are inducible for both erythroid and megakaryocytic antigens.

In a previous study we explored the inducibility of erythroid, granulocytic, monocytic and megakaryocytic antigens in the human multipotent leukemia cell line K562. In this study we examine the relationship between induction into the two lineages for which inducibility is most marked: erythroid and megakaryocytic types. Specifically, does any cell manifest markers of both lineages? We show that 1) cultured without inducer, a minority of single cells expresses both erythroid and megakaryocytic antigens, i.e., glycophorin A and Plt-1 antigen, 2) thymidine-hypoxanthine and phorbol dibutyrate each induce each antigen, 3) the percentage of individual cells carrying both antigens increases under each of these inducing conditions, 4) the induction of each is not only relative in terms of percentage of total cells that are positive, but also absolute in terms of mean antigen per cell and (taking into account cell multiplication) total antigen per ml of culture, and 5) the inducibility of individual cells bearing both erythroid and megakaryocytic markers is confirmed by using a different inducer (butyric acid) and antibodies for different inducer (butyric acid) and antibodies for different erythroid and megakaryocytic antigens (VIE-G4 and AP-3, respectively).

Induction of HL60 cell differentiation by tiazofurin and its analogues: characterization and efficacy.

Among inducers of myeloid differentiation for leukemic cells, tiazofurin is of special interest because its mechanism of action is known; it inhibits inosine monophosphate dehydrogenase and thus decreases the guanine nucleotide pool. Reported here are three aspects of tiazofurin induction of myeloid differentiation in HL60 human acute promyelocytic leukemia cells. First, inductive efficacy was evaluated for analogues ara-tiazofurin, xylo-tiazofurin, and selenazofurin, for dinucleotide anabolites thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD), and for a phosphodiesterase-resistant TAD analogue, beta-methylene TAD. The results showed that the parent compounds are more effective inducers than the dinucleotide derivatives and that the selenazole analogues are more effective inducers than the thiazole compounds. Second, HL60 cell induction by tiazofurin was shown to be synergistic with that produced by the antiviral agent ribavirin. Finally, tiazofurin was found to induce expression of a phosphatidylinositol-specific phospholipase C-sensitive Fc gamma-receptor III (FcRIII) on HL60 cells, a feature consistent with neutrophilic, but not monocytic, differentiation.

Erythroid differentiation of K562 cells: mixed colonies as an index of delayed expression of commitment.

K562 cells demonstrate commitment, defined as the clonal expression of a differentiated phenotype coupled with a limitation in proliferation. Upon exposure to certain agents, K562 cells are induced to synthesize hemoglobin, detectable by benzidine staining. If plated in semisolid medium, they produce benzidine-positive colonies, benzidine-negative colonies, and mixed colonies, the latter containing both positive and negative cells. To test whether or not mixed colonies represent a delay in the expression of commitment, we conducted two types of experiments. The first type showed that, following inducer removal, a delay in plating causes not only a decline in the number of mixed colonies, but also a rise in the proportion of negative colonies, with no change in the proportion of positive colonies. To explain this result, we propose that a plating delay can conceal a negative cell producing a positive cell if that cell division has occurred before plating. Instead of one mixed colony, one observes one positive colony and one other colony, either negative or mixed (depending on subsequent negative-to-positive events). Thus delay does not change the proportion of positive colonies, presumably because they breed true. But delay causes an increase in negative colonies to balance the decrease in mixed colonies due to concealment of negative-to-positive events and provides evidence that the converse, positive-to-negative events, do not occur. The second type of experiment utilized cordycepin, which inhibits commitment. We predicted that, if mixed colonies represent a delay in the expression of commitment, the addition of cordycepin to cells already exposed to thymidine should increase the percentage of mixed colonies. We found that cordycepin does indeed preferentially increase the proportion of mixed colonies. These two types of experiments provide evidence that mixed colonies represent a delay in expression of commitment. Such an inducible system, in which the commitment event and its expression can be separated in time by a generation or more, may provide an opportunity to more fully characterize the commitment process.

Induction of megakaryocytic characteristics in human leukemic cell line K562: polyploidy, inducers, and secretion of mitogenic activity.

Because of the rarity of megakaryocytes in the bone marrow, a cell line inducible for megakaryocytic characteristics provides a valuable model for study. When cultured with phorbol esters, the human multipotent hematopoietic leukemic cell line K562 can be induced to develop many megakaryocytic characteristics, viz. increased cell size, reduced growth rate, megakaryocytic antigens, and expression of the sis proto-oncogene, the structural gene for the B-chain of platelet-derived growth factor. Further aspects of this process are here presented. First, it induces the release of mitogenic activity into the medium. Second, phorbol dibutyrate induces polyploidy, a feature of normal megakaryocyte development. Third, mezerein and teleocidin, nonphorbol ester tumor promotors, also induce development of multinuclearity and polyploidy.

Induction of hemoglobin synthesis in K562 cells by carbon dioxide deficiency.

Proliferation and differentiation are inversely related in many cell culture systems. The study of inducible systems is facilitated by optimal growth conditions in order that whatever differentiation is observed may be attributed to a specific effect of the inducer, rather than to a nonspecific effect of adverse growth conditions. To investigate the role of CO2 supply in an inducible system, the K562 human leukemia cell line inducible for hemoglobin synthesis was studied at 10%, 5% and 1.5% CO2 concentrations. The lower the CO2 concentration, the higher the percentage of benzidine-positive cells but the slower the growth rate. This increase in benzidine positivity reflected hemoglobin synthesis as indicated by incorporation of 3H-leucine into globin chains. If, in addition to reducing CO2 concentration, the complete medium was replaced by a bicarbonate-free medium, the percentage of benzidine-positive cells was further increased and growth further slowed. However, if endogenously produced CO2 was retained by sealing the culture vessel, these effects were mitigated. Since addition of ribosides blocked these effects, the mechanism for these effects appears to be inhibition of riboside biosynthesis due to the depletion of CO2 as a substrate. The implication of this work is that, for reproducibility in studies of inducible systems in which reduction of proliferation may itself increase the probability of differentiation, the CO2 tension, the bicarbonate concentration in the medium and the rate of egress of endogenously produced CO2 must be kept constant.

Erythroid induction of K562 human leukemia cells: enhancement by purines.

K562 cells are human leukemia cells inducible for hemoglobin synthesis by a variety of agents. This report demonstrates that hypoxanthine, which alone has no inductive effect, enhances induction by thymidine, resulting in a greater absolute, as well as relative, percentage of benzidine positive cells. This effect is seen over a 20-fold concentration range for both thymidine and hypoxanthine. This enhancement involves commitment, i.e., a process in which the induction of hemoglobin synthesis is coupled to a limitation in the number of subsequent cell divisions. Although thymidine alone increases the percentage of cells in S phase, hypoxanthine does not augment this. Purines other than hypoxanthine also enhance induction by thymidine. This enhancement by hypoxanthine of thymidine induction is inhibited by pyrimidine nucleosides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, itself an effective K562 inducer, is not additive to thymidine and hypoxanthine, suggesting that hypoxanthine may act by reducing the supply of guanosine nucleosides.

K562 cell erythroid differentiation: requirement for a factor in fetal bovine serum.

K562 human erythroleukemia cells can be induced to make hemoglobin by a variety of inducing agents. Most of these agents are effective in media supplemented with fetal bovine serum (FBS), but not in media supplemented with newborn bovine serum (NBS). The active factor in FBS has an apparent molecular weight of 30,000 daltons and appears to be a protein on the basis of the following properties: lability at 100 degrees C, inactivation by desferrioxamine plus trypsin, resistance to periodate, and resistance to ribonuclease. Media containing NBS can be used for induction if supplemented by either this factor or transferrin of bovine or human origin. The small size of the active factor (mol. wt. approximately 30,000 daltons) indicates that it is not identical to bovine transferrin (mol. wt. approximately 77,000 daltons). However, when iron-saturated bovine transferrin is digested with trypsin, the peptide fragments produced resemble the FBS factor in activity, size, and reaction with antibovine serum transferrin.

K562 human erythroleukemia cells demonstrate commitment.

Commitment, ie, the decision to express a differentiated phenotype and to terminate proliferation irreversibly in the absence of inducer, was investigated in K562 human erythroleukemia cells. Cells were cultured for 0, 1, 2, 3, or 4 days with inducer and then plated in medium containing methylcellulose without inducer. Daily after plating, hemoglobin content was scored by benzidine staining, and growth was assessed by estimating the cell number per colony. With all inducers used, three types of colonies were found, those containing only benzidine-positive cells, those containing only benzidine-negative cells, and those containing both cell types (mixed colonies). Thymidine produced a progressive increase in the percentage of positive and mixed colonies and a progressive fall in the percentage of negative colonies. Whereas negative colonies grew at an exponential rate with a generation time of about 20 hours, positive colonies reached an average maximum size of 16 cells, representing a total of four divisions. Butyrate had a similar effect, except that the rise was greater for mixed colonies than for positive colonies, and the plateau in positive colony size was less evident. In contrast, CO2 depletion or hemin treatment induced an increase in the fraction of cells staining benzidine positive that was lost rapidly upon removal of the inducing condition. Thus, of the four conditions, thymidine and butyrate caused commitment, whereas hemin and CO2 depletion did not. Thus K562 cells, like Friend cells, demonstrate commitment, but, unlike Friend cells, demonstrate a significant rate of commitment in the absence of inducer and hence form a significant percentage of mixed colonies with or without inducer.

K562 human erythroleukemia cell variants resistant to growth inhibition by butyrate have deficient histone acetylation.

K562 is an established human erythroleukemia cell line, inducible for hemoglobin synthesis by a variety of compounds including n-butyrate. To elucidate the role of butyrate-induced histone acetylation in the regulation of gene expression in K562 cells, we isolated 20 variants resistant to the growth inhibitory effect of butyrate. Four variants having different degrees of resistance were selected for detailed study. All four were found to be resistant to the hemoglobin-inducing effect of butyrate, suggesting that the two aspects of butyrate response, restriction of growth and induction of hemoglobin synthesis, are coupled. Further, after (5 days) culture with butyrate, two of the four variants exhibit less acetylation of H3 and H4 histones than does the butyrate-treated parent. Analysis of histone deacetylases from the variants indicated that each variant was distinct and that butyrate resistance may be accounted for by decreased affinity of the variant enzymes for butyrate, increased affinity of the enzymes for acetylated histone, or both. The fact that variants selected for resistance to growth inhibition by butyrate are also deficient in butyrate-induced hemoglobin synthesis and have abnormal histone deacetylase activity argues for butyrate inducing K562 cells to synthesize hemoglobin and restrict growth via histone acetylation.

Hemin preferentially stimulates synthesis of alpha-globin in K562 human erythroleukemia cells.

K562 human erythroleukemia cells are an established cell line derived from an adult with chronic myelogenous leukemia. Hemin stimulates their synthesis of embryonic and fetal hemoglobins. We have found that their globin synthetic pattern depends on the concentration of added hemin. Clone RA6 was cultured with 0--100 microM hemin and the globin synthetic pattern determined by 3H-leucine incorporation and analysis of 3H-globins by polyacrylamide gel electrophoresis in Triton X acid urea followed by fluorography and densitometry. The higher the hemin concentration, the greater the synthetic rate of each type of globin. However, the relative increase was greatest for alpha-globin. We propose that the differential dependence of alpha synthesis on added hemin is a reflection of translational inefficiency of alpha messenger RNA and that this property is exposed when the translational capacity of the cell is limited by hemin deficiency. We suggest that the differential dependence of alpha-chain synthesis on added hemin in clone RA6 is evidence of an intrinsic deficiency in heme synthesis.

Erythropoiesis in vitro: enhancement by neuraminidase.

Neuraminidase treatment of human fetal liver or adult marrow cells prior to culture results in an increased number of erythroid colonies and bursts. No increase occurs in the number of nonerythroid colonies. The number of bursts having more than eight subunits is increased preferentially. Individual burst subunits are also enlarged. Neuraminidase-treated cells yield erythroid bursts when cultured in concentrations of erythropoietin insufficient to produce bursts from untreated cells. It is proposed that (1) neuraminidase treatment of adult and fetal cell mixtures specifically stimulates differentiation of erythroid precursors, (2) the preferential stimulation of erythroid bursts having many subunits suggests a preferential susceptibility of more primitive BFU-Es, and (3) neuraminidase treatment enhances the response of erythroid precursors to erythropoietin.

Inducers of erythroid differentiation in K562 human leukemia cells.

A variety of agents, including many known to induce erythroid differentiation in Friend murine erythroleukemia cells, were tested for their ability to induce erythroid differentiation in K562 humane erythroleukemia cells. Cells were grown in suspension culture and scored for erythroid differentiation by benzidine staining. Of 39 agents tested, 19 induced erythroid differentiation in K562 cells and 20 did not. A striking effect of the type of serum employed in the medium was observed. The majority of the agents inducing erythroid differentiation in medium containing fetal calf serum showed little activity in medium containing newborn calf serum.

Hemoglobin synthesis in cultures of hepatic erythroid cells from the human fetus.

A recent theory of the control of human fetal hemoglobin synthesis, based on studies in cultured adult marrow, proposes that the phenotypic expression of fetal hemoglobin is largely dependent on the level of differentiation of the parental stem cells; that is, the earlier the progenitor, the greater the ability of its progeny to express fetal hemoglobin [Papayannopoulou, Th., Brice, M. & Stamatoyannopoulos, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2923-2927]. To test this relationship with fetal tissue, we have studied hemoglobin synthesis in cultured human fetal liver, comparing gamma chain synthesis in the descendants of the early progenitors ("bursts") with that in the descendants of the later progenitors ("colonies"). Cells from the livers of midtrimester fetuses were cultured in methylcellulose with erythropoietin. The beta/(beta + gamma) globin synthetic ratio on days 5 to 7, when colonies predominated, was 0.09-0.11, a value characteristic of fetal reticulocytes, and on days 11 and 12, when bursts predominated, was 0.15-0.17. Thus, in fetal liver, the descendants of the earlier progenitor, the burst-forming unit, may be making more beta chains rather than more gamma chains, compared to descendants of the later progenitor, the colonyforming unit. Our data on fetal liver, taken together with the data on adult marrow by others, suggest that the erythroid colonies express the gene characteristic of the age of the organism to a greater degree than bursts, which express beta and gamma genes less specifically. Thus, the capacity for highly selective gene expression characteristic of differentiated cells appears to be less well developed in the burst-forming unit than in the colony-forming unit.

Erythroid colony formation from human fetal liver.

The liver of the human fetus, induced to abort by prostaglandin or by hypertonic saline, contains cells that form colonies in methylcellulose in vitro. The colonies are erythroid as identified by cellular staining of hemoglobin by benzidine. Colony formation is generally similar, with regard to number, size and time of development, to that observed in cultures of nonadherent cells from human adult marrow. The number of colonies observed increases with the concentration of erythropoietin used and with the concentration of cells plated and decreases with the time interval between intra-amniotic instillation of the inducing agent and culture. Colong number is not greatly influenced by fetal age in the period 16-20 weeks or by whether the inducing agent is prostaglandin or hypertonic saline. Prostaglandin- and hypertonic saline-induced abortuses thus provide an abundant source of human fetal erythroid tissue for morphologic and biochemical studies of erythroid development.