Kathleen M. Sabo

Identification of a human heme exporter that is essential for erythropoiesis

FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

Cytotoxicity/genotoxicity: The application of cell culture techniques to the measurement of marine sediment pollution

Abstract Fish cell cultures were used to determine whether in vitro aquatic animal cell culture systems were capable of detecting pollution in the marine environment. Cells derived from rainbow trout gonad (RTG-2) and bluegill fry tissues (BF-2) were used as model cell systems to measure cytotoxicity and genotoxicity following exposure to Puget Sound sediment extracts, benzo(a)pyrene and MNNG. Sediment was collected from several sites within Puget Sound known to be contaminated with compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorinated hydrocarbons and heavy metals. Each of the sediment samples was extracted with organic solvents and added to cultures of the two model cell systems in DMSO. Following exposure the cultures were evaluated for cell death, mitotic inhibition, stimulatory effects, and chromosomal damage. These cell cultures responded to the sediment extracts much as they did to known mutagenic/carcinogenic chemicals which were used as model compounds. Those sediments known to be contaminated, based on chemical analysis and historical use patterns were also found to be qualitatively and quantitatively more toxic than were sediments from areas which were relatively unaffected by human activity. The results show that in vitro cell systems are capable of detecting pollution in the marine environment and have the potential of being powerful tools in aquatic toxicology in conjunction with whole animals or as a method of screening materials prior to in vivo testing.

In vitro toxicity of eight mutagens/carcinogens for three fish ceil lines

Recent interest in and concern for the quality of the environment has prompted a great deal of research into methods of measuring and assessing changes in it. One problem of major interest is that of increasing amounts of mutagenic/carcinogenic chemicals generated by modern society and released into marine and freshwater ecosystems (KRAYBILL et ai. 1977). Numerous techniques involving whole animal testing for cancer, cell culture for mutation and cytogenetic changes and bacterial mutagenicity have been devised to assay specific chemicals and inspect unknown mixtures (KILBEY et ai. 1977). Little, however, has been done to determine the effects of foreign chemicals on aquatic organisms (DIAMOND AND CLARK 1970).

Longitudinal studies of telomere length in feline blood cells: Implications for hematopoietic stem cell turnover in vivo

OBJECTIVE To address questions about stem cell turnover in relation to telomere length dynamics, we analyzed telomere length in serial blood samples from cats. MATERIALS AND METHODS Lymphocytes and granulocytes from two newborn kittens, a 2-year-old cat, a 10-year-old recipient of a double autologous stem cell transplant, and a 10-year-old control animal were analyzed by fluorescence in situ hybridization and flow cytometry at 2-week intervals over a 1-year period. RESULTS At study onset, long telomeres were found in granulocytes and lymphocytes from the two kittens (mean +/- SD: 70.2 +/- 3.1 and 72.5 +/- 3.1 telomere fluorescence units [TFU], respectively) compared with the 2-year-old cat (55.6 +/- 2.5 and 64.1 +/- 4.3 TFU, respectively) and the two adult animals (49.6 +/- 1.5 and 45.4 +/- 0.8 TFU, respectively). The rate of telomere shortening in both granulocytes and lymphocytes was most rapid in the kittens (slope: -16.7 +/- 1.4 and -15.6 +/- 0.2 TFU/year, respectively). As in humans, telomere shortening with age was more rapid in lymphocytes than in granulocytes. An average rate of telomere attrition of -0.52 +/- 0.03 TFU per cell division was calculated for cultured lymphocytes from the two kittens, approximately 5-fold higher than the rate observed in human cells. CONCLUSIONS The average telomere length in cats is 5- to 10-fold longer than in humans, but the rate of telomere shortening is much higher both in vivo and in vitro. These observations are compatible with similar stem cell kinetics in both species.

The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

Expression from Second-Generation Feline Immunodeficiency Virus Vectors Is Impaired in Human Hematopoietic Cells

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

The Ex Vivo Expansion of Feline Marrow Cells Leads to Increased Numbers of BFU-E and CFU-GM but a Loss of Reconstituting Ability

Some studies in mice suggest that hematopoietic stem cells can be maintained and possibly expanded ex vivo. As there is a paucity of data from larger animals, we have studied hematologic reconstitution following autologous marrow transplantation in cats. Transplantation of very low density marrow cells (<1.050 g/ml), termed “1050 cells,” at 2 × 105 cells/kg leads to rapid hematopoietic recovery (granulocytes >200/μl by day 20 ± 2 and platelets >50 × 103/μl by day 21 ± 3). Recovery rates are comparable when 1‐2 × 107 nucleated marrow cells/kg are infused, suggesting that reconstituting cells are enriched 50‐ to 100‐fold in the 1050 cell preparation. To explore if the numbers of reconstituting cells could be expanded ex vivo, 1050 cells were cultured in the presence of 5 ng/ml recombinant human interleukin 1β, 10 ng/ml recombinant canine (rc)G‐CSF, 2 U/ml rHu erythropoietin, and 5 ng/ml rc stem cell factor. Maximum numbers of BFU‐E and colony‐forming units‐granulocyte/macrophage (CFU‐GM) were generated at day 6. However, when 106 1050 cells/kg (5× that needed for hematologic recovery) were cultured for six days and all resulting cells infused into irradiated donor animals, two of nine (22%) engrafted. Even when flt3 ligand (100 ng/ml) was added to cultures, only two of five animals (40%) engrafted (p = NS versus studies without flt3 ligand). These data confirm that BFU‐E and CFU‐GM provide inaccurate estimates of reconstituting cells and demonstrate that the number or function of feline reconstituting cells is impaired by in vitro culture with cytokines.

Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.

In Utero Transplantation of Monocytic Cells in Cats With α-Mannosidosis

Background. Lysosomal storage diseases are devastating illnesses, in large part because of their neurologic consequences. Because significant morbidity occurs prenatally, in utero (IU) therapy is an attractive therapeutic approach. Methods. We studied the feasibility and efficacy of IU injections of monocytic cells (derived from normal marrow) in feline &agr;-mannosidosis. Heterozygous cats were interbred to produce affected (homozygous) and control (heterozygous and wild-type) offspring. Thirty-seven pregnancies were studied in which fetuses were transplanted intraperitoneally (1×108 cells/kg recipient) at gestational days 27 to 33 and then each week for 2 weeks (term=63 days). After birth, affected kittens were evaluated clinically and pathologically, tissue &agr;-mannosidase levels were assayed, and in many studies, the numbers of &agr;-mannosidase-containing cells were enumerated. When male donor cells were transplanted into female recipients, engraftment was also quantified using polymerase chain reaction to amplify a Y chromosome-specific sequence. Results. We establish methods to transplant cats intraperitoneally while IU using ultrasound guidance, thus, describing a new large animal model for prenatal therapy. We show that the donor monocytic cells engraft and persist (for up to 125 days) in the brain, liver, and spleen, albeit at levels below those needed to alter the clinical or pathological progression of the &agr;-mannosidosis. Conclusions. This is the first study of monocyte transplantation in a large animal model of a lysosomal storage disorder and demonstrates its feasibility, safety, and promise. Delivering cells IU may be a useful strategy to prevent morbidities before a definitive therapy, such as hematopoietic stem-cell transplantation, can be administered after birth.

An all-feline retroviral packaging system for transduction of human cells

Abstract The subgroup C feline leukemia virus (FeLV-C) receptor FLVCR is a widely expressed 12-transmembrane domain transporter that exports cytoplasmic heme and is a promising target for retrovirus-mediated gene delivery. Previous studies demonstrated that FeLV-C pseudotype vectors were more efficient at targeting human hematopoietic stem cells than those pseudotyped with gibbon ape leukemia virus (GALV), and thus we developed an all FeLV-C-based packaging system, termed CatPac. CatPac is helper-virus free and can produce higher titer vectors than existing gammaretroviral packaging systems, including systems mixing Moloney murine leukemia virus (MoMLV) Gag-Pol and FeLV-C Env proteins. The vectors can be readily concentrated (>30-fold), refrozen (three to five times), and held on ice (>2 days) with little loss of titer. Furthermore, we demonstrate that CatPac pseudotype vectors efficiently target early CD34(+)CD38(-) stem/progenitor cells, monocytic and erythroid progenitors, activated T cells, mature macrophages, and cancer cell lines, suggesting utility for human cell and cell line transduction and possibly gene therapy.