Protein-protein interactions in hematology and phage display.

Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology. Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat. Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed. Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies. Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions. This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins. Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting. Phage-derived antibodies also facilitate analyses of the humoral antibody response. Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo. The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori. Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications. This paper focuses on current and future applications of antibody and epitope phage display technology in hematology.

Epitope mapping of neutralizing botulinum neurotoxin A antibodies by phage display.

Single-chain antibodies neutralize activity and bind nonoverlapping epitopes of botulinum A neurotoxin. Two phage display epitope libraries were constructed from the 1.3 kb of binding domain cDNA. The minimal epitopes selected against the single-chain Fv-Fc antibodies correspond to conformational epitopes with amino acid residues 1115 to 1223 (S25), 1131 to 1264 (3D12), and 889 to 1294 (C25).

DNA copy number alterations in HIV-positive and HIV-negative patients with diffuse large-cell lymphomas.

Individuals infected with HIV are at increased risk of developing aggressive non-Hodgkin's lymphoma with a worse prognosis than those similarly afflicted without HIV infection. The underlying genetic differences in tumor behavior between these two groups are not known. We explored the hypothesis that lymphomas from HIV-positive individuals have distinct somatic genetic changes that may provide clues to the genetic basis of disease progression and outcome. Genome-wide DNA copy number alterations (CNAs) in primary tumors from 14 HIV-positive and 11 HIV-negative patients with diffuse large B-cell lymphoma (DLCL) were quantified using comparative genomic hybridization (CGH). Tumors from HIV-positive patients displayed fewer regional DNA-CNAs than those from patients who did not have HIV. When CNAs were present, they occurred at lower frequency in HIV-positive patients. Gains at chromosomes 8q and Xp were the most frequent changes in the HIV-negative group, and gains on 2p and 12q were common in the combined HIV-positive and HIV-negative groups. No alteration was specific to AIDS-related DLCL. These data suggest that fewer somatic genomic changes are needed for progression to DLCL in HIV-immunocompromised hosts, and that other factors, such as reduced immune surveillance, may contribute to neoplastic progression.

Persistence of aneuploid immature/primitive hemopoietic sub-populations in mice 8 months after benzene exposure in vivo.

Benzene (bz) is a common environmental contaminant associated with increased risk of myeloid leukemia. Chronic bz exposure in vivo increases the frequency of aneuploid circulating lymphocytes in humans. However, there is no information about persistence of bz-associated aneuploidy in immature/primitive cells, at risk of leukemic transformation, after bz exposure in vivo. We explored the relationship between the induction and persistence of aneuploidy in primitive hemopoietic cells from mice that received oral doses of bz in vivo. Short- and long-term persistence of aneuploidy were evaluated in immature/primitive sub-populations (Lin(-)c-kit(+)Sca-1(+)), as well as lymphoid and myeloid cells, 6 days and 2-8 months after exposure. Mice receiving bz in a corn oil carrier, or corn oil alone, both have increased aneuploidy frequencies (1-5%, compared to <1% in untreated controls) in all sub-populations, 6 days after exposure. However, unlike bz-induced aneuploidy, corn oil-induced aneusomies are transient, with frequencies returning to background levels in lymphoid and myeloid cells, 9 weeks after exposure. The frequency (5-9%) of aneuploid lymphocytes and myeloid cells is higher at 9 weeks than at 6 days, suggesting that bz disrupts chromosomal segregation in differentiated cells and/or progenitors. About 8 months after bz exposure, the Lin(-)c-kit(+)Sca-1(+) sub-population contains up to 14% aneuploid cells with numerical chromosomal aberrations affecting chromosomes 2 or 11. These data demonstrate that bz induces DNA copy number changes in immature/primitive cells, and that these changes persist for long periods. Although, initial exposures are not leukemogenic, subsequent exposures of cells to genotoxins or oxidative radicals that induce additional genetic hits may increase the risk of transformation. The contribution of bz-induced aneuploidy in immature/primitive cells to leukemogenesis remains to be determined.

Dermal benzene and trichloroethylene induce aneuploidy in immature hematopoietic subpopulations in vivo.

Accumulation of genetic damage in long-lived cell populations with proliferative capacity is implicated in tumorigenesis. Hematopoietic stem cells (hsc) maintain lifetime hematopoiesis, and recent studies demonstrate that hsc in leukemic patients are cytogenetically aberrant. We postulated that exposure to agents associated with increased leukemia risk would induce genomic changes in cells in the hsc compartment. Aneusomy involving chromosomes 2 and 11 in sorted hsc (Lin(-)c-kit(+)Sca-1(+)) and maturing lymphoid and myeloid cells from mice that received topical doses of benzene (bz) or trichloroethylene (TCE) was quantified using fluorescence in situ hybridization. Six days after bz or TCE exposure, aneuploid cells in the hsc compartment increase four- to eightfold in a dose- and schedule-independent manner. Aneuploid lymphoid and myeloid cells from bz- and TCE-treated mice approximate controls, except after repeated benzene exposures. Aneuploid cells are more frequent in the hsc compartment than in mature hematopoietic subpopulations. Hematotoxicity was also quantified in bz- and TCE-exposed hematopoietic subpopulations using two colony-forming assays: CFU-GM (colony-forming units/granulocyte-macrophage progenitors) and CAFC (cobblestone area-forming cells). Data indicate that bz is transiently cytotoxic (< or =1 week) to hsc subpopulations, and induces more persistent toxicity (>2 weeks) in maturing, committed progenitor subpopulations. TCE is not hematotoxic at the doses applied. In conclusion, we provide direct evidence for induction of aneuploidy in cells in the hsc compartment by topical exposure to bz and TCE. Disruption of genomic integrity and/or toxicity in hsc subpopulations may be one step in leukemic progression.

Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis.

This report describes the development and validation of quantitative microsatellite analysis (QuMA) for rapid measurement of relative DNA sequence copy number. In QuMA, the copy number of a test locus relative to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. Use of simple sequence repeats is advantageous because of the large numbers that are mapped precisely. In addition, all markers are informative because QuMA does not require that they be polymorphic. The utility of QuMA is demonstrated in assessment of the extent of deletions of chromosome 2 in leukemias arising in radiation-sensitive inbred SJL mice and in analysis of the association of increased copy number of the putative oncogene ZNF217 with reduced survival duration in ovarian cancer patients.

Radiation-induced translocations in mice: persistence, chromosome specificity, and influence of genetic background.

The translocation frequency response in the chromosomes of peripheral blood lymphocytes is widely used for radiation biomonitoring and dose estimation. However, this assay is based upon several assumptions that have not been rigorously tested. It is typically assumed that the translocation frequency in blood lymphocytes reflects the level of genomic damage in other hemopoietic tissues and is independent of the chromosome probe and genetic background. We conducted studies to evaluate these assumptions using mice with different genetic backgrounds. Six different whole-chromosome fluorescence in situ hybridization (FISH) probes were used to detect translocations in peripheral blood lymphocytes at multiple times after whole-body irradiation. Translocation frequencies were chromosome-independent at 6 and 16 weeks after exposure but were chromosome-dependent at 1. 5 years after exposure. Similar translocation frequencies were observed in blood, bone marrow and spleen at 1.5 years, supporting previous suggestions that genetically aberrant peripheral blood lymphocytes may derive from precursor populations in hemopoietic tissues. Translocations measured 66 h after irradiation differed among some strains. We conclude that the translocation frequency response is a complex phenotype that is influenced not only by exposure dose but also by genetic background, the choice of chromosome analyzed, and time after exposure. These results raise important considerations for the use of the FISH-based translocation frequency response for radiation dosimetry and biomonitoring.

Comparative genomic analyses of primary effusion lymphoma.

BACKGROUND: A rare subset of human immunodeficiency virus (HIV) lymphomas, known as primary effusion lymphomas (PELs), are high-grade tumors carrying human herpes virus 8. Mechanisms postulated to contribute to lymphomagenesis include impaired immune surveillance, alterations in hemopoietic regulatory pathways due to expressed viral genes, and acquisition of genomic alterations in regions of the genome that contain regulatory genes. In PEL, limited information exists about the nature of genome-wide aberrations in these rare lymphomas. METHODS: We used comparative genomic hybridization to detect regions of sequence gain and loss throughout the genome of 8 PEL cases. Regions of DNA sequence loss or gain were confirmed using forward and reverse hybridization and t-statistic analyses. RESULTS: Genomic aberrations were identified in 6 of 8 cases, including recurrent gain of sequence in chromosomes 12 [ish enh (12q22;12q23, 12q12;12q23)] in 3 of 8 cases and X [ish enh (X, Xp)] in 2 of 8 cases. CONCLUSIONS: DNA copy number changes occurred in a majority of PEL cases and are consistent with changes observed in other HIV lymphomas. These observations suggest that common genetic events may occur in HIV-associated lymphoid malignancies, but they probably do not contribute to the unique markers and morphology of PEL. Although individual genetic loci have been evaluated previously in a few PEL cases, to our knowledge this study represents the first reported genome-wide scan of copy number changes in these rare HIV-associated tumors.

Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations.

BACKGROUND: Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function. Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants. We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur. METHODS: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry. Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood. The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry. RESULTS: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein. The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells. CONCLUSIONS: These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells. This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations.

Cell cycle regulatory gene abnormalities are important determinants of leukemogenesis and disease biology in adult acute lymphoblastic leukemia.

To test the hypothesis that cell cycle regulatory gene abnormalities are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a Southwest Oncology Group protocol for abnormalities of the genes, retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant expression occurred in 33 (85%) patients in the following frequencies: Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had abnormalities in 2 or more genes. Outcomes were compared in patients with 0 to 1 abnormality versus patients with multiple abnormalities. The 2 groups did not differ in a large number of clinical and laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%, respectively). Patients with 0 to 1 abnormality had a median survival time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months (n = 13; 95% CI, 4-12 months) for those with multiple abnormalities (P <.01). Stem cells (CD34+lin-) were isolated from adult ALL bone marrows and tested for p16(INK4A) expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and sorted stem cells lacked p16(INK4A) expression. In 2 other patients only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16 expression was present in the CD34+ lin- compartment in 95% (median) of 9 patients whose lymphoblasts expressed p16(INK4A). Therefore, cell cycle regulatory gene abnormalities are frequently present in adult ALL lymphoblasts, and they may be important determinants of disease outcome. The presence of these abnormalities in the stem compartment suggests that they contribute to leukemogenesis. Eradication of the stem cell subset harboring these abnormalities may be important to achieve cure.

Low frequency epithelial cells in bone marrow aspirates from prostate carcinoma patients are cytogenetically aberrant.

BACKGROUND: Low frequency epithelial cells occur in bone marrow aspirates of 25-50% of patients with locally confined prostate carcinoma. It is assumed that bone marrow epithelial cells derive from the primary tumor; however, it has not been established unequivocally that they are tumor cells. Immunofluorescence approaches were used to quantify the frequency of epithelial cells in bone marrow aspirates from prostate carcinoma patients and genotypic analyses were used to determine whether they contained numeric aberrations of chromosomes 1, 7, and 8. METHODS: Epithelial cells in bone marrow aspirates collected after radical prostatectomy were visualized using fluorescence microscopy and fluorophore-linked antibodies against cytokeratin 8,18 (CK) and prostate specific antigen (PSA). Antibodies specific for proliferating nuclear cell antigen (PCNA) were used to evaluate the cycling status of discriminated cells. Copies of chromosomes 1, 7, and 8 in the discriminated epithelial cells were quantified using fluorescence in situ hybridization. RESULTS: CK+ cells were present in bone marrow aspirates from 30 of 66 patients (approximately 45%) at a median frequency of 1.4 CK+ cells/10(5) mononuclear cells. Few CK+ epithelial cells in the bone marrow aspirates coexpressed PSA and none of the CK+ cells expressed PCNA. Approximately 70-75% of the CK+ cells contained 7 and 8 aneusomies. Gains of chromosome 1 occurred in 42% of the CK+ cells. CONCLUSIONS: The majority of CK+ cells in bone marrow aspirates collected after surgery are cytogenetically aberrant, which is consistent with a primary tumor origin. The prevalence and frequency of CK+ cells is independent of tumor stage/grade and androgen treatment.

Cytogenetically aberrant cells are present in the CD34+CD33-38-19- marrow compartment in children with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL), the most common cancer in childhood, is characterized by clonal proliferation of transformed lymphoblasts that comprise the majority of marrow and/or blood specimens. Although the leukemic cells typically express antigens associated with lymphoid maturation or activation (ie CD19, CD38, etc), it has been suggested that ALL blasts may evolve from a more primitive precursor. Increased understanding of the phenotypic and molecular heterogeneity of cells in ALL may provide clues to leukemogenesis and/ or impact prognostication or treatment. We utilized a phenotype/genotype approach to measure the prevalence and frequency of cytogenetically aberrant cells in a phenotypically defined primitive compartment (CD34+33-19-38-; CD34+Lin-). Bone marrow cells were flow cytometrically sorted into CD34-Lin+, CD34+Lin+ and CD34+Lin- subpopulations. Fluorescence in situ hybridization (FISH) was used to quantify the frequency of cells with aneusomies in the sorted populations. Approximately 26% (5/19) of ALL cases at diagnosis contain cytogenetically aberrant CD34+Lin- cells. The frequency of cytogenetically aberrant cells in the CD34+Lin- compartment is independent of FAB, WBC and blast counts. These data indicate that cytogenetically aberrant cells may reside in a phenotypically defined primitive subpopulation and suggest that ALL blasts in some patients may evolve from a precursor compartment.

Rescue from lethal irradiation correlates with transplantation of 10-20 CFU-S-day 12.

Repopulation by donor cells of a bone marrow ablated by irradiation is now recognized to proceed in two phases: initial repopulation that may be temporary followed by permanent engraftment of longterm repopulating cells (LTRC). While a single LTRC has been shown to be capable of restoring the entire lymph-hemopoietic system of an irradiated animal, the identity of the temporary repopulating cells has not been established unequivocally. We used the results of transplantation of subpopulations successively enriched for LTRC and containing varying numbers of CFU-S-12 (colony-forming units in the spleen at day 12 post transplantation) and progenitors to determine the likely cell type and number of cells needed for initial survival after radiation. Subpopulations from untreated and 5-fluorouracil-treated mice were discriminated on the basis of antibody reactivity, Hoechst 33342 and rhodamine 123 fluorescence intensity and light scattering properties. The minimum rescue inocula varied greatly in CFU-GEMM, BFU-E and CFU-GM content. One to two CFU-S-12 were uniformly present in all isolated suspensions that rescued 50% of lethally irradiated animals. In view of the known average seeding efficiency of CFU-S, our studies suggest that transfusion of 10-20 CFU-S day 12/13 is responsible for radioprotection. Evidence that multiple CFU-S day 12/13 are needed for initial repopulation is also supported by quantitative estimates of the number of mature cells that can be produced by CFU-S. Transfusion of a single CFU-S day 12/13 can be shown to be grossly inadequate to provide the number of peripheral blood cells needed to ameliorate the severe pancytopenia following lethal irradiation by day 12-14. Our data also indicate that 5-fluorouracil-treated marrow subpopulations appear inferior to untreated subpopulations in their ability to contribute to initial repopulation when transfused at low cell doses into lethally irradiated recipients.

Molecular cytogenetic abnormalities in multiple myeloma and plasma cell leukemia measured using comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to identify recurrent regions of DNA sequence loss and gain in 21 multiple myeloma (MM) and plasma cell leukemia (PCL) primary tumor specimens and cell lines. Multiple regions of non-random sequence loss and gain were observed in 8/8 primary advanced stage tumors and 13/13 cell lines. Identification of sequence copy number changes was facilitated by statistical analyses that reduce subjectivity associated with identification of copy number changes and by requiring that sequence changes are visible using both red- and green-labeled tumor DNA. Loss of sequence on 13q and 14q and gain of sequence on 1q and chromosome 7 occurred in 50-60% of the population. In general, cell lines carry more and larger regions of sequence gain and loss than primary tumors. Regions of sequence copy number change that recur among MM cell lines and primary tumors include, in order of prevalence, enh(1q12qter), dim(13), enh(7), enh(3q22q29), enh(11q13.3qter), dim(14q11.2q31), enh(8q21qter), enh(3p25pter), dim(17p11.2p13), and dim(6q22.1q23). Population distributions of genome-wide changes in primary tumors reveal "hot-spots" of sequence loss from 13q12.1-q21, 13q32-q34, 14q11.2-q13, and 14q23-q31. Genomic changes detected using CGH are consistent with those identified using banding analyses, although recurrent involvement of additional regions of the genome are also evident. A higher prevalence of genomic changes is visible using CGH compared to banding. Identification of recurrent regions of sequence gain and loss provides opportunities to identify regions of the genome that may be involved in the malignant phenotype and/or disease progression.

Molecular cytogenetic analysis of cytokeratin 20-labeled cells in primary tumors and bone marrow aspirates from colorectal carcinoma patients.

BACKGROUND: Low frequency epithelial cells in bone marrow from colorectal carcinoma patients are associated with an increased risk of recurrence and decreased survival. Current immunohistochemical approaches to detect epithelial cells in bone marrow aspirates rely on antibodies against cytokeratin 18 (CK18). The predictive value of CK18-based detection strategies is limited by false-positives that occur in approximately 30% of cases. Cross-reactivity of anti-CK18 antibodies with nontumor cells may contribute to the false-positive rate. Cytokeratin 20 (CK20) shows more restricted expression than CK18 and labels cells in colorectal tumors. METHODS: Immunofluorescence assays were used to quantify CK20-labeled cells in bone marrow aspirates and tumors from 18 Dukes stage C and D colorectal carcinoma patients to determine whether CK20 is useful in detecting micrometastases. Fluorescent in situ hybridization was used to determine whether CK-labeled subpopulations carried genomic aberrations associated with colorectal carcinoma. RESULTS: CK20-labeled cells occurred at frequencies approximately 5 x 10(-5) in control bone marrow aspirates from patients without colorectal carcinomas. Approximately 10(-4) CK20-labeled cells were present in 4 of 11 bone marrow aspirates (45%) from patients with Dukes stage D colon carcinoma. The mean frequency (5 x 10(-5) of CK20-labeled cells in Dukes stage C and D rectal carcinoma patients was statistically similar to control values. CONCLUSIONS. A subset of CK20-labeled cells in primary tumors and hepatic metastases are aneusomic. CK20-labeled cells in bone marrow aspirates are cytogenetically normal. These data demonstrate that CK20 cells in solid tumors may be cytogenetically aberrant, but suggest caution in the use of CK20 to detect micrometastases in bone marrow aspirates.

Genotype/phenotype analyses of low frequency tumor cells using computerize image microscopy.

Techniques to identify low frequency (i.e., 10(-4)-10(-5)) tumor cells in bone marrow and peripheral blood of cancer patients provide opportunities for early detection of disseminated disease and characterization of the properties of cells released from the tumor. Low frequency epithelial cells in marrow and blood can be detected using immunophenotypic markers directed against intracellular and/or cell surface antigens. However, nonspecific phenotypic labeling may compromise the ability to discriminate tumor from nontumor cells. We describe optimization of slide-based approaches that facilitate identification and subsequent molecular cytogenetic characterization of rare tumor cell populations in hematopoietic tissues. Colon tumor cells seeded in a hematopoietic background provided a model system to optimize methodologies that are applicable to detection and quantification of micrometastases in clinical specimens. Mixtures of cytogenetically aberrant epithelial cells and hematopoietic cells on slides were labeled with an anti-cytokeratin 20 (anti-CK20) antibody that recognizes > 90% of colon adenocarcinomas. Computerized image analysis was used to record the location of the immunofluorescent cells on microscopic slides. Cells on slides were then hybridized using fluorescence in situ hybridization (FISH) with repeat-sequence DNA probes to detect aneusomies. Previously discriminated epithelial cells were relocated for molecular cytogenetic characterization. Tumor cells present at frequencies approximating 5 x 10(-5) were discriminated on the basis of immunofluorescence and cell size. A low frequency (4 x 10(-4)) population of normal hematopoietic cells also labeled with anti-CK20 (data not shown). This strategy of sequential immunophenotyping and molecular cytogenetic analyses may be useful to discriminate tumor from nontumor cells in cancer patients with micrometastatic disease.

Cytogenetically aberrant cells in the stem cell compartment (CD34+lin-) in acute myeloid leukemia.

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.

Unusual kinetics of white cell clearance in transfused mice.

BACKGROUND: Donor white cells (WBCs) in blood transfusions are responsible for complications in recipients, including alloimmunization, graft-versus-host disease (GVHD), and virus transmission and reactivation. The recent use of sequence-specific polymerase chain reaction assays to monitor the kinetics of clearance of donor WBCs in transfused humans and dogs found transient recirculation of donor lymphocytes on Days 3 to 5 after transfusion; this presumably reflected an abortive GVHD reaction to major histocompatibility complex-incompatible recipient cells, after which donor WBCs were cleared to undetectable levels. STUDY DESIGN AND METHODS: This study sought to develop a murine model to further characterize the kinetics and major histocompatibility complex restriction of donor WBC clearance. A sensitive murine Y chromosome-specific polymerase chain reaction assay was developed and applied to serial blood samples collected after transfusions of allogeneic blood to naive inbred, primed inbred, and outbred mice, as well as after transfusions of gamma-radiated blood to naive inbred mice. RESULTS: In inbred mice, both naive and primed to the allogeneic blood donor, transfused WBCs were not cleared to undetectable levels for more than 1 month after transfusion. Transfused outbred mice also showed prolonged donor WBC survival, although at lower levels than inbred mice. There was no evidence of GVHD in either inbred or outbred mice, and gamma radiation had no significant impact on donor WBC persistence. CONCLUSION: These results contrast with the rapid clearance of donor WBCs observed in humans and dogs. The immunologic basis for this discrepancy remains unclear. Caution should be exercised in any extrapolation to humans of conclusions drawn from results in transfused mice.

Intracellular dynamics of c-myc mRNA traffic in single cells in situ.

Processing and intracellular transport of RNA transcripts are essential for gene expression. Translational regulation of gene expression may occur by several mechanisms, including control of transcript movement from sites of synthesis to site(s) of translation. We describe temporal analysis of the intracellular translocation of c-myc transcripts from the site of synthesis in the nucleus to sites of translation in the cytoplasm. Fluorescence in situ hybridization (FISH) and quantitative fluorescence microscopy were used to measure intracellular traffic of c-myc transcripts in individual recombinant cells following activation of c-myc sequences linked to a heat shock promoter. C-myc nuclear transcripts are visible in the nucleus within minutes of heat exposure. Transcripts remain in the nucleus for at least 4 hr after gene activation. Transport of c-myc transcripts to the cytoplasm begins approximately 1 hr after cells are returned to 37 degrees C. These data demonstrate the feasibility of measuring intracellular transcript transport following gene activation and provide a description of the kinetics of intracellular traffic of inducible c-myc transcripts in heated cells in situ.