Propolypeptide of von Willebrand factor circulates in blood and is identical to von Willebrand antigen II.

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.

Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile.

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.

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.

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.

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.

Monoclonal antibodies to human progesterone receptor: characterization by biochemical and immunohistochemical techniques.

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.

Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta.

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.

Zyn-Linker delivery of antirheumatic agents.

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.

Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry.

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.

Kinetics of infected cell appearance as a determinant of number of human immunodeficiency virus-1 infectious units.

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.

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.

K562 human leukemia cell passages differ in embryonic globin gene expression.

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.

Opioid growth factor regulates the cell cycle of human neoplasias.

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.

A novel drug delivery system using IL-2 activated NK cells and Zyn-linked doxorubicin.

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.

Circulating killer cells in women undergoing needle-directed excisional breast biopsy.

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.

Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice.

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.

Isolation of UV-sensitive clones from a haploid frog cell line.

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.

Monoclonal antibody to a protein of the nucleus and mitotic spindle of mammalian cells. Localization and synthesis throughout the cell cycle.

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.

Tropism of human immunodeficiency virus 1 isolates for H9 cells and U937 cells.

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.