Kenneth S. Landreth

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP‐16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL‐2 expression by VP‐16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF‐7, MDA‐MB‐435S, and MDA‐MB‐A231 following in vitro treatment with 50–100 μM VP‐16. Elevation of BAX protein expression in the presence of VP‐16 alone did not correlate with reduced viability or induction of apoptosis in MCF‐7, MDA‐MB‐435S, or MDA‐MB‐A231. VP‐16 did effectively block the breast cancer cell lines evaluated (MCF‐7 and MDA‐MB‐435S) at G2/M phase of the cell cycle, confirming activity of the drug in vitro. MCF‐7 and MDA‐MB‐435S cells that were pre‐treated with VP‐16 and subsequently exposed to 1.0–12.0 μg/m1 4‐hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP‐16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP‐16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL‐2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.

Regulation of Eosinophilopoiesis in a Murine Model of Asthma

Eosinophilic inflammation plays a key role in tissue damage that characterizes asthma. Eosinophils are produced in bone marrow and recent observations in both mice and humans suggest that allergen exposure results in increased output of eosinophils from hemopoietic tissue in individuals with asthma. However, specific mechanisms that alter eosinophilopoiesis in this disease are poorly understood. The current study used a well-characterized murine animal model of asthma to evaluate alterations of eosinophil and eosinophil progenitor cells (CFU-eo) in mice during initial sensitization to allergen and to determine whether observed changes in either cell population were regulated by T lymphocytes. Following the first intranasal installation of OVA, we observed sequential temporal elevation of eosinophils in bone marrow, blood, and lung. In immunocompetent BALB/c mice, elevation of bone marrow eosinophils was accompanied by transient depletion of CFU-eo in that tissue. CFU-eo rebounded to elevated numbers before returning to normal baseline values following intranasal OVA exposure. In T cell-deficient BALB/c nude (BALB/cnu/nu) mice, CFU-eo were markedly elevated following allergen sensitization, in the absence of bone marrow or peripheral blood eosinophilia. These data suggest that eosinophilia of asthma results from alterations in two distinct hemopoietic regulatory mechanisms. Elevation of eosinophil progenitor cells in the bone marrow is T cell independent and likely results from altered bone marrow stromal cell function. Differentiation of eosinophil progenitor cells and phenotypic eosinophilia is T cell dependent and does not occur in athymic nude mice exposed to intranasal allergen.

5-Hydroxyindoleacetic acid and substance P profiles in patients receiving emetogenic chemotherapy.

Background. Even though direct cause and effect has not been proved, clinical evidence suggests serotonin and substance P (SP) are involved in the emetic response following chemotherapy. Because of several parallels, we hypothesized that SP release, like serotonin, may be propagated by chemotherapy and both substances can be measured in biological fluids, and correlated with a particular phase of emesis. Methods. Urinary 5-hydroxyindoleacetic acid (5-HIAA) was assessed by HPLC; serum and urine SP were measured by immunoassay. In addition to construction of neurotransmitter profiles, all SP data were grouped according to cisplatin dosages, = or>75 mg/m 2 versus <75 mg/m2, and phase of emesis, acute versus delayed. Analyses of these data were performed by repeated measures analysis of variance. Results. Samples were collected over a 72-hour period from 26 adult patients who received cisplatin-(n=13) or non-cisplatin-containing (n=13) chemotherapy. Mean baseline 5-HIAA: creatinine ratios were 5.23 and 5.16 in females and males, respectively; mean baseline SP levels were 392 and 181 pg/mL in females and males, respectively. Comparisons between SP data stratified by cisplatin dosage and emetic phase were significantly different, P <0.0001. Conclusions. Laboratory studies provide additional evidence that serotonin and SP are involved primarily, though not exclusively, in acute and delayed vomiting, respectively.

Gender-Specific Effects of Prenatal Chlordane Exposure on Myeloid Cell Development

Work previously reported by this laboratory indicated that prenatal chlordane exposure affected macrophage function in young adult mice. Because these macrophage effects were due to exposure during the development of the immune system, the possibility of a persistent effect on the development of myeloid stem and progenitor cells was considered. Female mice were treated with either 0 or 8 mg of chlordane per kilogram body weight daily for 18 days during pregnancy. Myeloid hemopoietic activity of bone marrow cells from 6-week-old offspring was evaluated for in vitro colony-forming units-in-culture in response to exogeneously added recombinant forms of the cytokines granulocyte/macrophage-colony stimulating factor, macrophage-CSF, and interleukin 3 (IL-3). There was a significant depression of the numbers of bone marrow colony forming units-granulocyte/macrophage (CFU-GM), CFU-IL-3, and CFU-macrophage (CFU-M) in only the female offspring. Male offspring consistently demonstrated no difference in the CFU-GM, CFU-IL-3, or CFU-M. Prenatal treatment with chlordane did not significantly affect the number of recoverable viable bone marrow cells in either male or female mice.

Selective Myelotoxicity of Propanil

Propanil, a commonly used herbicide, has been previously shown to be immunotoxic for selected immune functions as well as specific cell types, such as the macrophage. Propanil has also been shown to cause a methemoglobulinemia and anemia through direct action on the erythrocyte. Demonstrated toxicity to both macrophages and erythrocytes raised concern for the possible myelotoxicity of propanil which could contribute to the observed effects of exposure. Therefore, the effect of propanil on several stem and progenitor cell types was assessed 7 days after acute propanil exposure. The results described herein show that propanil, at doses of 50-200 mg/kg body wt, resulted in reduction in the number of myeloid stem cells and early myeloid and erythroid progenitor cells. No reduction in the numbers of more differentiated myeloid and erythroid progenitor cells was noted at even the highest dose used (200 mg/kg). In addition, no statistically significant difference in number of leukocytes per femur was noted. These data suggest that propanil is myelotoxic to early hemapoietic stem cells, but that this reduction is apparently compensated by proliferation of more differentiated progenitor cells for the myeloid and erythroid lineages. It remains unknown whether chronic exposure leads to progressive depletion of additional myeloid and erythroid cells.

Effects of the protein tyrosine phosphatase CD45 on FcγRIIa signaling and neutrophil function

OBJECTIVE Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. METHODS The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. RESULTS Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. CONCLUSIONS These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.

Hematopoiesis: The Role of Stromal Integrin Interactions in Pro-B Cell Proliferation.

Recent studies using long term bone marrow cultures have concluded that adherence of lymphoid precursors to the underlying stromal cells is required for normal B cell development. However, the function of specific integrin interactions in B cell development remains unresolved. In our laboratory, we observed that maximal proliferation of pro-B cells required the presence of stromal cells and that stromal cytokines alone could not replace the requirement for stromal cell contact. For that reason, we questioned whether integrin interactions play a role in regulating pro-B cell proliferation in the bone marrow. Murine pro-B cell line C1.92 expressed VLA-4, CD44, and fibronectin-receptor. Abrogation of binding of these molecules to stromal cell ligands using blocking antibodies resulted in failure of pro-B cell adherence and significant decreases in pro-B cell proliferation. Disruption of single integrin interactions did not compromise either adhesion of pro-B cells to stromal cells or IL-7 stimulated proliferation. Taken together, these data suggest that normal pro-B cells interact with stromal cells through multiple integrin interactions and that integrin mediated potentiation of pro-B cell proliferation is functionally redundant and not affected by failure of single ligand interactions.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice

BACKGROUND Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. OBJECTIVE To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. METHODS The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. RESULTS BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. CONCLUSIONS Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

Isolation and Characterization of a Stromal Cell Line

Considerable progress in identifying cells and cytokines that regulate primary B cell differentiation has been made in the last few years, and it is now possible to present a model that identifies the stages at which various developmental signals act on B lineage cells. The formulation of such a scheme has been possible largely because of the use of various in vitro systems that allow the stromal cell dependent growth of B cells and their precursors and the application of molecular techniques to the analysis of cells in these cultures (Dorshkind 1990; Kincade et al. 1989).