1987 American Society of Human Genetics Social Issues Committee Report.
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Genetics, Medical↗
Biomedical subjects
P T Rowley
Publications and source records attributed to P T Rowley.
Genetic discrimination: rights and responsibilities of tester and testee: summary of a workshop sponsored by the Social Issues Committee, American Society of Human Genetics, November 2, 1986.
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Confidentiality↗
Pregnant women identified as hemoglobinopathy carriers by prenatal screening want genetic counseling and use information provided.
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Female↗
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.
Carbon Dioxide↗
Oncogene expression in myelopoiesis.
Oncogenes are a class of genes hypothesized to be causally related to neoplasia. To date, specific oncogenes have been recognized chiefly by their ability to transform test cells to a neoplastic phenotype. This has been accomplished largely through mutational analysis of the genotype of retroviruses or through the analysis of tumor cell DNA by in vitro transfection of rodent fibroblasts. Oncogenes are believed to arise by some genetic alteration from normal cellular genes called proto-oncogenes. Although the normal function of most proto-oncogenes is unknown, it has been proposed that they may function as tissue-specific and temporally specific regulators of differentiation. The role of oncogenes in lymphoid malignancies has been extensively analyzed. Less is known about their role in myeloid leukemias and especially in normal myelopoiesis. Space limitations permit discussion of only salient features of a limited number of oncogenes; we have arbitrarily selected myc, myb, fos, fms, fes, sis, and abl.
Animals↗
Multipotent human hematopoietic cell line K562: lineage-specific constitutive and inducible antigens.
K562 cells have been reported to display a variety of non-erythroid properties. Using 28 lineage-specific monoclonal antibodies, we analysed which antigens are present spontaneously and which are inducible by a variety of agents. The data suggest that (1) antigens of a given lineage are preferentially responsive to certain inducers, e.g. megakaryocytic antigens to phorbol ester, and (2) a given inducer may influence antigens of different lineages in opposite directions, e.g. phorbol dibutyrate, not only induces megakaryocytic antigens, but also decreases granulocyte and erythroid antigens. We conclude that the K562 cell, despite its malignant origin, retains some capacity for expression of alternative programs of differentiation, a characteristic of the normal multipotent hematopoietic stem cell.
Antigens↗
Regulation of the activity of heme degradative enzymes in K562 erythroleukemic cells: induction by thymidine.
Heme oxygenase is rate-limiting in the heme degradative pathway, and its activity is induced by a host of chemicals. In K562 human erythroleukemic cells, heme oxygenase activity was not increased by exposure to potent inducers, such as cobalt chloride, bromobenzene, and heme. Indeed heme treatment severely suppressed the enzyme activity, and at 18 h the activity measured less than 5% of the control. Heme and cobalt chloride did not inhibit activities of NADPH-cytochrome c (P-450) reductase and biliverdin reductase to a marked degree. In contrast, treatment of cells with thymidine/hypoxanthine alone, or in combination with cobalt chloride, caused an increase in the activity of three enzymes of heme degradation. It is suggested that with thymidine, which is a committing inducer of hemoglobin synthesis, the induction of activity of the three enzymes of the heme degradation pathway is coupled with cell differentiation. On the other hand, in the case of heme, a noncommitting inducer of hemoglobin synthesis, induction of hemoglobin synthesis and increase in heme degradation activity may be independent.
Bromobenzenes↗
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.
Cell Differentiation↗
Oncogene expression in neurofibromatosis.
To investigate the role of oncogenes in malignancies characteristic of neurofibromatosis, oncogene transcripts were quantitated in a neurofibrosarcoma and in control tissue from a patient with hereditary neurofibromatosis. Sis and N-ras were moderately hyperexpressed, raf, Blym, and erbA were slightly hyperexpressed, and abl, erbB, fes/fps, fgr, fos, mos, myb, myc, N-myc, rasHarvey, rasKirsten, ros, src, and yes were not hyperexpressed in the tumor compared to the control tissue. Although additional tumors will be assayed before conclusions are possible, it may be significant that the two oncogenes most hyperexpressed are prior suspects for a pathogenetic role in tumors of the nervous system.
Gene Expression Regulation↗
Genetic counseling of asymptomatic carriers in a primary care setting. The effectiveness of screening and counseling for beta-thalassemia trait.
In a prospective, controlled, stratified, experimental effectiveness study of screening and counseling, 25 000 consecutive adults were screened for thalassemia trait. Eight hundred forty-three adults had a mean corpuscular volume less than 77 fL; 192 (22% of those with microcytosis) had hemoglobin A2 greater than 3.5%, proving beta-thalassemia trait. Video-program, neutral-educational, and patient-centered counseling methods produced equal levels of learning, retention, psychologic impact, and effects on life adjustment immediately and at 2 and 10 months after counseling. Ninety-nine percent of the patients told other persons about the counseling, and 43% had 106 others screened. Factors related to having someone else screened included plans to have children (p less than 0.002), being younger (p less than 0.0025), better education (p less than 0.05), and having high knowledge of thalassemia (p = 0.05). For maximum effectiveness, screening and counseling programs should focus on patients for whom a positive result has high significance.
Adolescent↗
The contribution of red cell morphology to the diagnosis of beta-thalassemia trait.
The contribution of red cell morphology to the diagnosis of beta-thalassemia trait in a predominantly healthy population was analyzed. The smears from 59 beta-thalassemia trait subjects already identified by DEAE-cellulose microcolumn chromatography, 60 subjects with other forms of microcytosis, and 64 nonmicrocytic subjects were randomly selected and evaluated blind. The "experimental" morphologic criteria were the presence of both basophilic stippling and either microcytosis or target cells. The "control" morphologic criteria were the presence of both microcytosis and poikilocytosis. Although the sensitivity of both sets of criteria were only moderate (73%), the specificity and predictive value of the experimental criteria were very high (99% and 98% respectively).
Erythrocytes, Abnormal↗
Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells.
Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.
Cell Cycle↗
Regulation of K562 cell transferrin receptors by exogenous iron.
Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.
Animals↗
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.
Adenine↗
Toward providing parents the option of avoiding the birth of the first child with Cooley's anemia: response to hemoglobinopathy screening and counseling during pregnancy.
Will the development of safe, accurate prenatal diagnosis for Cooley's anemia result in a reduction in the unwanted birth of children with Cooley's anemia in the United States? Since detection of couples at risk only through the birth of an affected child will lead to a minimal reduction in the incidence of the disease, carrier screening will be necessary. A pilot project of prenatal hemoglobinopathy screening is being conducted in Rochester, New York, to answer the question "Should hemoglobinopathy carrier screening and genetic counseling of positives be part of routine prenatal care?" In the first 27 months of this study, 6,641 unselected pregnant women have been screened on the first prenatal visit, regardless of race and ethnic origin. Of these, 304 have been positive for some type of hemoglobinopathy. Of the 293 analyzed here, the proportion coming for counseling was 61% of the total group and 77% of the thalassemia trait subgroup. Of this number, the proportion wanting their mate tested was 98% of the total group and 100% of the thalassemia trait subgroup. The proportion of those counseled whose mate was actually tested was 60% for the total group and 70% for the thalassemia trait subgroup. The proportion of at risk couples wanting amniocentesis (mostly for detection of sickle cell disease) was 61%. We conclude that, when comprehensive hemoglobinopathy screening is incorporated into routine prenatal screening, the majority of positive women make an extra visit to receive an explanation, nearly all women coming for counseling want their mate tested, the majority of mates come for testing, and the majority of couples at risk want amniocentesis. Further, pregnant women with beta-thalassemia trait may be more inclined to act upon identification as a carrier than are positive women at large.
Abortion, Therapeutic↗
Demonstration of commitment by K562 human erythroleukemia cells.
Commitment, i.e. 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 following plating, hemoglobin content was scored by benzidine staining and growth was assessed by estimating 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 benzidine positive and benzidine negative cells (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 which 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.
Benzidines↗
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.
Butyrates↗
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.
Animals↗