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Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677 nm), as an alternative and/or complementary probe to PKH26 and CFSE(1) for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0-96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm-658 nm) excitation capabilities.

The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue Lavender, CellVue Plum and CellVue NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.

We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon (near infrared), NeuroVue Red and NeuroVue Green. Using pair-wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue Maroon (excitation maximum 647 nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP-eGFP transgene, we demonstrate that either NeuroVue Maroon or NeuroVue Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Adoptively transferred IL-2 activated NK (A-NK) cells selectively accumulate within tumor metastases which recommends them as vehicles for locoregional drug delivery. Zyn-Linkers are membrane-binding lipophilic dyes which can be coupled by a variety of conjugation chemistries to therapeutic agents. We have previously demonstrated that A-NK cells labeled with PKH26 are able to accumulate within established B16 melanoma pulmonary metastases by 16 h at a concentration of over 600 cells/mm2 of tumor tissue (Basse et al. J. Exp. Med. 174: 479 1991). Zyn-205 is a prodrug in which doxorubicin is attached to a similar Zyn-Linker through an acid-sensitive bond. We have optimized the ex vivo labeling conditions and found that a 10 min incubation with 25 microM Zyn-205 results in the uptake of over 10(8) drug molecules per cell with no effect on either cell viability or cytolytic activity up to 24 h after labeling. Given these parameters, the amount of drug which may be carried to and concentrated in metastatic lesions represents a local concentration of approximately 15 microM. In addition, A-NK cells carrying Zyn-Linked doxorubicin at an equivalent dose of 25 micrograms/kg was therapeutically comparable to a systemic dose of 8 mg/kg (320x more) in the 3LL model of experimental metastasis. These data indicate that A-NK cells bearing Zyn-Linked chemotherapeutic agents represent a unique and feasible method to target chemotherapeutic agents to cancer metastases and that therapeutic doses can be attained without unwanted systemic exposure.

The identification of prognostic variables is an important aspect of managing and counseling women with endometrial adenocarcinoma. The surgical stage, age, cell type, depth of myometrial invasion, and histologic grade have all been previously demonstrated to be related to prognosis. Several reports have indicated that tumor ploidy as determined by flow cytometry with fresh or fixed cells removed from paraffin blocks of endometrial adenocarcinomas can contribute to the assessment of prognosis. To verify the significance of DNA content in endometrial adenocarcinoma, we conducted an historical cohort study on a subgroup of women from a Gynecologic Oncology Group (GOG) protocol of early clinical stage disease. Flow cytometry was performed at one facility on cells extracted from blocks obtained from several GOG member institutions. Blocks were submitted for 293 of 933 eligible patients. Ninety-two histograms were of good quality, with 55 interpreted as diploid and 37 as aneuploid. One hundred sixty-two histograms were technically suboptimal, of which 137 were considered probably diploid, 13 probably aneuploid, and 12 unacceptable due to high background noise. Of the commonly accepted prognostic variables, only depth of invasion was significantly related to the ploidy status. There was no discernable difference in survival between patients with diploid and patients with probable diploid and probable aneuploid tumor types. Incorporation of the flow cytometry data into a proportional hazards regression model adjusted for age and surgical stage revealed a significant increased risk of disease-related death (relative risk, 4.1; 95% confidence interval, 2.3 to 7.3) for patients with aneuploid tumor type as compared to patients with diploid tumor type. This study confirms the prognostic significance of ploidy determination by flow cytometry and also indicates some of the difficulties of retrospectively applying this technology to cooperative group studies.

Needle-directed excisional breast biopsy (NDBB) is performed on many women having suspicious mammograms. Only a small subset of these women (approximately 20%) will be found to have breast cancer. The goal of this study was to test the hypothesis that presence (or absence) of circulating lymphocytes able to kill cultured breast cancer-derived cell lines would discriminate the subset of women with early forms of breast cancer from those with benign breast disease. The immune status of over 200 women undergoing NDBB was ascertained at the time of biopsy from a peripheral blood sample. Use of a standard surface immunophenotyping panel able to discriminate a variety of both natural killer cells and cytotoxic T lymphocytes revealed no significant differences when compared to a group of healthy volunteers or to the normal ranges established for our laboratory. Functional assays (cytotoxicity studies) were performed in a blinded fashion on peripheral blood lymphocytes from a subset (N = 31) of these women using a panel of target cell lines. Target cells included the NK sensitive K562; the NK resistant Daudi; and two breast cancer-derived cell lines, the hormone sensitive MCF7 and MDA-MB-435 which has been shown to metastasize in immunocompromised mice. No differences were apparent among the groups with respect to ability to kill either K562 or Daudi. All individuals undergoing NDBB had an enhanced ability to kill the breast cancer-derived cell lines compared to healthy donors. Individuals with breast cancer killed, on average, twice as many MCF7 cells as than did individuals with benign breast disease whereas both groups killed approximately three times as many MDA-MB-435 cells as did healthy donors. Further, a higher proportion of the women undergoing NDBB (2-3x) had relatively high cytotoxic ability directed against the breast cancer-derived lines than was found among healthy donors. These data expand earlier observations (Hamilton et al., 1988) that women with stage I breast cancer had elevated ability to kill MCF7 to include a substantial subset of all women with suspicious mammograms.

Despite our increasing ability to manage rheumatoid arthritis through systemic medication, refractory joints require local administration of more aggressive therapy in a substantial number of patients. These studies tested whether a new class of molecules designated Zyn-Linkers could deliver and retain therapeutics in a joint. Zyn-Linkers are synthetic lipid-like molecules designed to insert into cell membranes and enhance drug delivery to cells. After intra-articular injection into the knee of NZW rabbits, Zyn-Linkers bound rapidly and homogenously to synovial lining cells. Chelating Zyn-Linkers which contained Re-186 or Y-90 were synthesized to evaluate localization and retention after intra-articular injection. Initial studies using Re-186 Zyn-Linker gave excellent localization as evaluated by whole-body imaging: counts in the knee region represented > 90% of counts present in the whole body for at least 4-6 days postinjection. Similar results were obtained using a Y-90 Zyn-Linker and this agent was used for biodistribution studies due to its greater stability and ease of preparation. Efficacy and safety of Y-90 Zyn-Linker as a potential radiation synovectomy agent were estimated by extrapolation of biodistribution data to humans. A therapeutically effective dose of 8,000 cGy to synovium was calculated to require intra-articular injection of 3.4 mCi Y-90 Zyn-Linker, a value less than or equal to doses of particulate Y-90 agents used clinically in Europe. The predicted safety profile for Y-90 Zyn-Linker was excellent, with estimated doses to nontarget organs and tissues falling well within FDA-recommended safety levels for research-only radiopharmaceuticals. In addition to exhibiting desirable localization and retention properties, Zyn-Linkers may also be synthesized to release antirheumatic drugs such as methotrexate at controlled rates. This suggests substantial potential for these drug delivery molecules as chemical synovectomy agents which may be used concurrently with systemic chemotherapy to improve management of refractory joints.

Asymptomatic individuals seropositive for human immunodeficiency virus (HIV) progress in a heterogeneous fashion toward AIDS. To facilitate monitoring of disease progression and response to therapy, a rapid, new flow cytometric assay (FCA) lymphocyte p24-FCA, has been devised to quantify peripheral blood lymphocytes expressing cell-associated HIV-1 p24 antigen. Results from 55 asymptomatic, HIV-1-seropositive, serum p24 antigen-negative individuals ranged from undetectable (less than 0.1%) to 13.6% p24+ lymphocytes (mean, 2.0%). Mean values for three other groups studied were 0.1% for seronegative, viral culture-negative laboratory workers (n = 24); 4.2% for untreated patients with AIDS (n = 16); and 0.3% for AIDS patients receiving zidovudine (n = 11). Lymphocyte p24-FCA values were inversely related to the number of days to positive viral cultures and to levels of CD4+ lymphocytes. The ratio of p24+ lymphocytes to CD4+ lymphocytes may reflect the fraction of infected CD4+ lymphocytes. Lymphocyte p24-FCA determination may provide a method for monitoring response to antiretroviral therapy regardless of serum p24 antigen status.

Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line. [35S]Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules. Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells. In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation. Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action. These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.

Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus-1 (HIV-1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV-1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8-10 d, harvested, and evaluated for evidence of HIV-1 infection by the presence of cell-associated HIV-1 antigens and/or by the presence of particle-associated reverse transcriptase activity in cell culture supernatants. Thirty-minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV-1-infected H9 cells by greater than 99.99%. To the same degree, a multi-step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV-1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 micrograms/ml was ineffective in reducing HIV-1 infectivity.

Human immunodeficiency virus 1 (HIV-1) produced in the human T lymphoblastoid H9 cell line infected cells of that line more readily than cells of the human monocytoid U937 line. While both cell lines expressed detectable levels of the CD4 molecule on their surfaces, the H9 and U937 cell lines differed in expression of major histocompatibility complex class I and class II antigens. Both H9 and U937 cells were infected initially with HIV-1 derived from H9 cells. Cell-free culture supernatants were harvested after the cells had been infected for at least 1 month. Culture supernatant from HIV-infected H9 cells was used to infect H9 and U937 cells. Conversely, culture supernatant from HIV-infected U937 cells was used to infect H9 and U937 cells. The percentages of cells infected at each of several time points during the first few days after infection were determined by flow cytometric analysis of cell-associated HIV-1 major core protein p24. Infection of each cell line was more efficient when the cell type infected was identical to that in which the infecting supernatant was produced. However, this difference in tropism was not generated early after infection of each cell line, as might have been expected if this effect were mediated by cell surface molecules acquired during the process of budding through the cell membrane.

Cell cycle parameters of two cold-sensitive Chinese hamster cell lines have been determined by flow cytometric analysis of cell populations stained with the DNA specific fluorochrome mithramycin. The most striking finding is a depletion of S phase cells, detectable as early as 12 h after a shift to the nonpermissive temperature of 30.0 degrees C and complete by 24 h following the shift. There is a substantial increase in the proportion of cells in the G1 phase of the cell cycle compared to wild type cells under identical conditions but, surprisingly, the proportion of cells having a G2/M DNA content is quite similar in the two populations. The proportion of tetraploid cells present in these populations is not sufficient to account for this observation. Reversibility of the cold-induced block was tested by returning cells held for three days at the nonpermissive temperature to the permissive temperature. Cells having a G1 content of DNA do reenter the S phase, beginning approximately 8 h after a return to the permissive temperature.

Nuclear envelopes of somatic cells have at least two different major proteins in the 60-70(X 10(3] molecular weight range (lamins A(C) and B) that seem to be involved in chromatin attachment. In contrast, nuclear envelopes from clam germinal vesicles have only a single major protein of the same size class (approximately 67 X 10(3) Mr) and have no chromatin attached to them. The data presented in this report show that this 67 X 10(3) Mr clam protein shares a variety of physical properties with lamins A(C) and B, derived from rat liver nuclei. These properties include similar size, although different isoelectric points; phosphorylated forms; strong tendencies to cross-link by disulphide bonds; presence of carbohydrates, demonstrated by direct incorporation of mannose and labelling with borohydride; and shared epitopes, demonstrated using both monoclonal and polyclonal antibodies. Taken together, these observations identify the clam 67 X 10(3) Mr protein, the major structural protein of a nuclear envelope that lacks attached chromatin, as being lamin-like and demonstrate that it is more closely related to lamin A(C) than to lamin B.

An isolation procedure has been developed which yielded five clones of haploid frog cells which are sensitive to ultraviolet light. This procedure employed a conventional mutagenesis, followed by time for phenotypic expression and then an enrichment for UV-sensitive mutants. The enrichment relies upon the uptake of bromodeoxyuridine (BrdU) by repairing cells following UV-induced damage, rendering repair-proficient cells differentially sensitive to photolysis by black light. The photolysis is potentiated by use of the bisbenzimidazole dye Hoechst 33258. The enriched population was screened for radiation-sensitive isolates resulting in 5 sensitives from 96 tested. No mutants were obtained from 300 isolates tested from a population which had not undergone enrichment.

Variants of the Chinese hamster ovary cell line CHO-KI (ATTCCCL61)which grow almost normally at 38.5C but very poorly or not at all at 30C were obtained after treatment with mutagens and application of an indirect selection procedure. Two kinds of variants were recovered. In the first of these, the cold sensitive phenotypw is expressed completely only at low cell densities. At higher cell desity, growth continues at the nonpermissive temperature, but at a reduced rate. In the second class, the cold snesitive phenotype is independent of cell density. Two members of the latter class were studied in detail. In both lines, after shift to the nonpermissive temperature, the rateof H-thymidine incorporation declines markedly; the rates of H-uridine and H-phenylalanine uptake are less drastically reduced. Autoradiographs indicate that the decline in thymidine uptake at the nonpermissive temperature is due to an elongation of part of the cell cycle, so that a smaller proportion of the cells lie in the synthetic (S) phase of the cell cycle with a consequent reduction in the fraction labeled cells. The uridine labeling patterns of the mutants appear to rule out a ribsomal lesion. Low temperature growth inhibition of both cell strains was reversible. In one of the cell lines, an apparent stretching of the cells at the low temperature produces substantial alterations in cell shape.

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.

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 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.

The generally mild bleeding disorder of von Willebrand disease is associated with abnormalities of two distinct plasma proteins, the large multimeric von Willebrand factor (vWF), which mediates platelet adhesion, and von Willebrand antigen II (vW AgII), which is of unknown function. The two proteins were found to have a common biosynthetic origin in endothelial cells and megakaryocytes, which explains their simultaneous absence in the severe form of this hereditary disease. Shared amino acid sequences from a 100-kilodalton plasma glycoprotein and from vW AgII are identical to amino acid sequences predicted from a complementary DNA clone encoding the 5' end of vWF. In addition, these proteins have identical molecular weights and immunologic cross reactivities. Monoclonal antibodies prepared against both proteins recognize epitopes on the pro-vWF subunit and on a 100-kilodalton protein that are not present on the mature vWF subunit in endothelial cell lysates. In contrast, polyclonal antibodies against vWF recognize both pro-vWF and vWF subunits. Thus, the 100-kilodalton plasma glycoprotein and vW AgII are identical proteins and represent an extremely large propolypeptide that is first cleaved from pro-vWF during intracellular processing and then released into plasma.

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.

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.

Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10(-4) following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.

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.

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.

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.

K562 is a human leukemia cell line inducible by a variety of agents for the synthesis of embryonic and fetal hemoglobins. We compared early and late passages to determine whether a change has occurred in globin synthetic pattern. Clone LA4, derived from passage 199 which had been frozen by Lozzio in 1973, was compared with clone RA6, derived from a line received from Rutherford in 1979. Globin synthetic pattern was determined by incubation with [3]leucine, separation of globins by Triton-X100 polyacrylamide gel electrophoresis, and analysis by fluorography. For RA6, hemin-induced synthesis was greatest for zeta globin but minimal for epsilon globin, whereas for LA4 it was greatest for epsilon globin but minimal for zeta globin. Both lines are pseudotriploid with three No. 11 and three No. 16 chromosomes. However only RA6 has a translocation involving the short arm of chromosome 11 which contains the locus of the beta globin gene cluster. However, translocation-associated deletion does not simply explain the deficient inducibility of epsilon synthesis because G gamma and A gamma globins, whose genes are linked to the epsilon gene, are similarly inducible in the two lines.

The metaphase chromosome transfer system of McBride and Ozer (1973) has been adapted to a haploid, euploid, frog cell line. Genes coding for a deoxypyrimidine kinase and an enzyme responsible for a thymidine-specific saturable transport system have each been transferred at frequencies between 10(-6) and 10(-5) transferents per cell treated. Revertants for each of these two genes were observed at frequencies between 10(-8) and 10(-7) revertants per cell tested. Selfing controls showed no transferents. Two colonies were obtained in which cotransfer of both genes may have occurred. Activities of the transferred genes were assayed by incorporation of [3H]thymidine into alkali-stable, acid-precipitable material. Growth properties of 13 transferents in various media were also determined and presence of the appropriate enzymes inferred. These transferents were tested for stability early (25 generations) after transfer and were found to be stable. All 13 transferents possess the normal haploid number of chromosomes (n = 13) with no cytologically detectable chromosomal fragments.

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.

We report here the isolation of a monoclonal antibody, J17, that reacts with a conserved vertebrate protein antigen that is present in the spindle apparatus during mitosis but found within the nucleus during interphase. Immunofluorescence microscopy demonstrates that the J17 antigen is found in numerous punctate regions that are distinct from nucleoli. Furthermore, this antigen is not directly associated with kinetochores, the nuclear envelope, or with metaphase chromosomes. Antibody J17 immunoprecipitates a single polypeptide of very high molecular weight (over 250 000) from K562 human erythroleukemia cells pulse-labeled with 14C-leucine. This polypeptide is converted quantitatively to a stable 220-kilodalton product within one cellular generation. We discuss the possible relevance of this processing event for transport into the nucleus. The J17 antigen is synthesized throughout the cell cycle in Chinese hamster ovary cells.

In order to optimize detection of human immunodeficiency virus-1 (HIV-1)-infected cells, the temporal appearance of virus antigens in newly infected H9 cell cultures was examined. Analyses were accomplished by indirect immunofluorescence labeling with each of 10 monoclonal antibodies and evaluation by flow cytometry. Of the antibodies examined, those specific for HIV-1 capsid protein p24, matrix protein p17, or their precursor molecule p55 allowed the earliest and most sensitive detection in infected cells fixed to allow detection of intracellular antigen. Discrimination of infected cells from uninfected cells was much less sensitive when three antibodies specific for HIV-1 glycoproteins were used to detect intracellular or cell surface antigen. In several experiments involving the time course of infection, we observed no differences in cell numbers between infected and uninfected H9 cultures initiated at identical cell concentrations. We hypothesized that it might be possible to quantitate infectious HIV-1 virions from the kinetics of infected cell appearance. Straight-line relationships between the log p24-positive cells and the time after infection were observed. These quantitative observations were employed to calculate the number of infectious units originally added to the culture that were capable of infecting H9 cells. The production of infectious virus, but not of cytopathic effects, was required. The results of this novel approach to the titration of infectious HIV-1 particles agreed well with those from median cell culture infective dose determination. This method could be employed with other infectious agents for which detection of cell-associated antigens is possible in cell cultures not destroyed by infection.

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.

The effect of different population densities of mice per cage on plasma corticosterone, peripheral lymphocytes and specific lymphocyte subpopulations was investigated. The animals were housed in groups of 2, 4 or 8 mice per cage and the blood samples were taken from each animal of these groups on days one, 7 and 14. A significant elevation (P less than 0.05) in plasma corticosterone concentration was observed in the group of 8 mice per cage on days one and 7 as compared with those of 2 or 4 mice per cage. The number of peripheral lymphocytes was significantly decreased in the groups of 2 (P less than 0.01) and 8 (P less than 0.05) mice per cage as compared with the group of 4 mice per cage on day one. A significantly decreased number of lymphocytes (P less than 0.01) in the group of 8 mice per cage continued to day 7. There were no significant differences in specific lymphocyte subpopulations observed among these groups. The results of this study suggest that a population density of 4 mice per cage induced minimal stress compared to that induced by the population densities of 2 or 8 mice per cage. Since stress is known to induce alteration in a variety of biological functions, the population density of mice per cage should be considered in the interpretation of research data.

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.

Progesterone receptor (PR) from a human endometrial carcinoma (EnCa 101) grown in nude mice consists of two hormone-binding proteins with mol wt around 116,000 and 85,000. To generate monoclonal antibodies against this receptor, PR was partially purified from EnCa 101 and used to immunize Robertsonian mice. Immune mouse spleens were fused with HL-1 Friendly myeloma-653 cells, and hybridomas were screened by solid phase dot-blot assay and double antibody precipitation. Seven stable hybridomas were obtained, designated hPRa 1-7. Subisotyping revealed that hPRa 1 and 6 were immunoglobulin G2b, while the remainder were immunoglobulin G1. Ultracentrifugation in high salt sucrose gradients showed that six of the seven antibodies effected a shift of [3H]progestin-labeled PR from EnCa 101; only hPRa 4 was ineffective in this regard. Protein blots of EnCa 101 cytosols and DEAE eluates revealed that hPRa 1, 3, 4, 5, and 7 recognized both PR proteins equally. hPRa 2 recognized principally the 116,000 mol wt PR protein; it recognized the lower mol wt PR protein very poorly if at all, whereas hPRa 6 recognized only the 116,000 mol wt protein. Interestingly, the latter was consistently detected as a closely migrating triplet. Immunolocalization of PR by hPRa 1-7 in tissue sections was confined to nuclei of target tissues and varied in intensity: hPRa 7 greater than 3 = 5 greater than 6 = 2 greater than 1 greater than 4. In proliferative phase uterus, the intensity of staining was ranked: endometrial gland nuclei (3+) greater than myometrial cell nuclei (2-3+) greater than endometrial stromal cell nuclei (0-1+). Thus, seven monoclonal antibodies directed against human PR have been prepared, and their suitability for the study of PR by biochemical and immunohistochemical techniques has been demonstrated.

One possible mechanism explaining the action of interferon (IFN) on squamous cell carcinoma (SqCC) of the head and neck is the modulation of major histocompatibility antigen expression on tumor cells. We tested the ability of gamma interferon (gamma-IFN) to modulate major histocompatibility class I antigens and beta 2-microglobulin (beta 2-M) on two carcinoma cell lines derived from SqCC of the head and neck. Major histocompatibility class I antigens and beta 2-M were detected using a two-step immunochemical stain; antigen expression was quantified using flow cytometry. gamma-IFN increased constitutive antigen expression by as much as five times on both cell lines. Maximum modulation was seen within 72 hours of exposure to gamma-IFN at clinically attainable doses (10 U/mL to 100 U/mL). The presence of gamma-IFN in cell cultures was necessary for continued modulation of surface antigens. These findings suggest a possible mechanism of action and encourage further clinical trials with gamma-IFN.