Analysis of human megakaryocytic cells using dual-color immunofluorescence labeling.

BACKGROUND: Megakaryocytes are classically identified by their cellular morphology and expression of platelet glycoproteins. METHODS: In this study, the expression of GPIIIa (CD61) on hemopoietic cells was analyzed by dual-fluorescence flow cytometry. RESULTS: All monocytic cells (CD14+) were shown to coexpress CD61. As the expression of platelet protein on these monocytic cells cannot be reduced by treating the cells with anticoagulant (ethlyenediaminetetraacetic acid [EDTA]), this observation is not simply due to platelet adhesion. When sorted CD61(lo)CD14+ cells were studied by light and electron microscopy, platelets or platelet fragments could not be detected on the cell surface. These cells were found to have typical monocytic morphology but no megakaryocytic characteristics. CONCLUSIONS: This finding demonstrates that without careful definition, the quantitation of megakaryocytic cells will be inappropriately high. A clear and unambiguous criteria for the identification of megakaryocytic cells is described based on the high expression of platelet glycoprotein (e.g., CD61(hi) or CD41(hi)) but not the monocyte marker (CD14(neg)).

Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status.

The most primitive hematopoietic stem cells capable of longterm reconstitution of the entire hematopoietic system following transplantation are characterized by their ability to exclude both Rhodamine 123 and Hoechst 33342 dyes (Rh/Ho(dull)), and are an appropriate target population for the determination of stem cell ultrastructure. We have used a fluorescence-activated cell sorter to enrich to near purity these rare, highly quiescent cells. Analysis of the in vitro growth characteristics of Rh/Ho(dull) cells demonstrated an obligatory requirement for multiple growth factors, with 62% of the sorted population having the capacity to form colonies in the presence of CSF-1 + IL-1alpha + IL-3 + SCF. The Rh/Ho(dull) cells were small, with profiles having a mean diameter of 4.6 microm. Ultrastructural examination showed numerous ribosomes and several mitochondria in the thin rim of cytoplasm surrounding the nucleus, with other cytoplasmic organelles revealed in serial sections. The cells were generally homogeneous in appearance apart from the nucleus, which had an irregular shape with a single deep indentation. The heterochromatin around the margin was distinctly more pronounced in about 50% of nuclei. The findings provide a basis for studying the structural changes that occur with progressive differentiation of early hematopoietic cells.

A NIMA homologue promotes chromatin condensation in fission yeast.

Entry into mitosis requires p34(cdc2), which activates downstream mitotic events through phosphorylation of key target proteins. In Aspergillus nidulans, the NIMA protein kinase has been identified as a potential downstream target and plays a role in regulating chromatin condensation at mitosis. nimA- mutants arrest in a state that physically resembles interphase even though p34(cdc2) is fully active. Despite evidence for the existence of NIMA-like activities in a variety of cell types, the only bona fide NIMA homologue that has been identified is the nim-1 gene of Neurospora crassa. We report here the isolation of a fission yeast NIMA homologue, and have designated this gene fin1 and the 83 kDa predicted protein p83(fin1). Overexpression of fin1 promotes premature chromatin condensation from any point in the cell cycle independently of p34(cdc2) function. Like NIMA, p83(fin1) levels fluctuate through the cell cycle, peaking in mitosis and levels are greatly elevated by removal of C-terminal PEST sequences. Deletion of fin1 results in viable but elongated cells, indicative of a cell cycle delay. Genetic analysis has placed this delay in G2 but, unlike in nimA mutants of Aspergillus, p34(cdc2) activation appears to be delayed. Interaction of fin1 mutants with other strains defective in chromatin organisation also support the hypothesis of p83(fin1) playing a role in this process at the onset of mitosis. These data indicate that NIMA-related kinases may be a general feature of the cell cycle and chromatin organisation at mitosis.

Enhancement of radiation-induced regrowth delay by gemcitabine in a human tumor xenograft model.

Gemcitabine, a cytidine nucleoside analogue, has schedule-dependent antitumor activity in vitro and in vivo. Gemcitabine also has dose- and time-dependent radiosensitization properties in vitro. Thus it may have therapeutic application in combination with radiation. The aims of this study were to investigate whether gemcitabine could enhance radiation-induced tumor regrowth delay in a human squamous carcinoma (FaDu) xenograft in nude mice and to examine the effect of gemcitabine on radiation-induced apoptosis in in vivo tumors. Radiation was given locally to the tumors twice daily in 2 Gy fractions over 2 weeks for 5 days/week. Significant regrowth delay enhancement was observed which was dependent on gemcitabine schedule. Effective schedules using maximum tolerated gemcitabine doses were twice weekly and once weekly, but not daily. Significant toxicity occurred with radiation plus twice weekly gemcitabine, but enhancement was seen using gemcitabine doses well below the maximum tolerated dose. Both gemcitabine and radiation led to apoptotic cell death, but this was not increased when both treatments were combined. These results indicate that gemcitabine may be of therapeutic value as a radiation enhancer in the treatment of human cancers. Preliminary studies suggest that increased apoptotic cell death is not a mechanism leading to this enhancement.

Expression of the tumor-associated mucin MUC1 in an ovarian tumor cell line.

Epithelial sialomucins constitute a family of high-molecular-weight glycoproteins associated with epithelial cell surfaces. Aberrant expression of these molecules has been observed in certain types of human epithelial tumors. Members of the MUC1 family of mucins isolated from different tissue types have been shown to differ in biochemical properties and in immunological reactivity. The polymerase chain reaction (PCR) has been used in a study of the MUC1 mRNA expressed in ovarian tumor cells. The intron/exon structure of the ovarian mucin gene has been examined, the nucleotide sequence of regions of the cDNA 5' and 3' to a central highly repetitive region of the molecule determined and genomic clones for this mucin from the ovarian tumor cell line COLO316 have been isolated and analyzed. The results are compared with the nucleotide sequence data obtained by others for MUC1 cDNA in breast and pancreatic cell lines. With a single nucleotide exception, the splicing pattern and nucleotide sequence obtained from MUC1 mRNA in the ovarian cell line is the same as that of the mRNA found in breast and pancreatic cell lines. However, it appears that the use of alternate splice acceptors for intron I in the ovarian cell line studied here is independent of the specific sequence variation thought to determine splicing of MUC1 in breast tumor cells.