Rushdia Z. Yusuf

Direct measurement of local oxygen concentration in the bone marrow of live animals

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

The Lkb1 metabolic sensor maintains haematopoietic stem cell survival

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Paclitaxel Resistance: Molecular Mechanisms and Pharmacologic Manipulation

It has been approximately ten years since the Food and Drug Administration (FDA) approved paclitaxel for the treatment of platinum resistant epithelial ovarian carcinoma. Since the approval, the drug has found therapeutic applications in a variety of schedules and in a wide variety of epithelial malignancies. Its novel mechanism of action provided the hope that it would demonstrate anti-neoplastic activity in multidrug resistant tumor cells. Unfortunately, as with other chemotherapeutic drugs, resistance is commonly seen. Laboratory investigation has defined a wide variety of resistance mechanisms including overexpression of multidrug resistance (MDR-1) gene, molecular changes in the target molecule (betatubulin), changes in apoptotic regulatory and mitosis checkpoint proteins, and more recently changes in lipid composition and potentially the overexpression of interleukin 6 (IL-6). This review describes the in vitro molecular data that define and support the various mechanisms of resistance and critically evaluates the evidence for the participation of these mechanisms in clinically relevant paclitaxel resistance. This review also explores pharmacologic attempts to modulate paclitaxel resistance, principally through inhibition of the MDR-1 drug efflux pump. Future avenues for drug resistance research and its pharmacologic manipulation in the clinic are discussed.

Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

Description of paclitaxel resistance-associated genes in ovarian and breast cancer cell lines

PurposeTo identify genes involved in the paclitaxel resistance phenotype.MethodsHigh-density Affymetrix HG-U95Av2 microarrays were used to quantify gene expression in the resulting cell lines, SKOV-3TR, OVCAR8TR and MCF-7TR, and their drug-sensitive parental lines, SKOV-3, OVCAR8 and MCF-7.ResultsThree paclitaxel-resistant human ovarian and breast cancer cell lines were established. We identified 790 (SKOV-3TR), 689 (OVCAR8TR) and 964 (MCF-7TR) transcripts that were more than twofold overexpressed relative to their expression in the corresponding parental cell line. A comparison of these transcripts identified eight genes that were significantly overexpressed in all three drug-resistant daughter cell lines. These genes included MDR1, a gene often implicated in both in vitro and in vivo resistance to multiple chemotherapeutics, including paclitaxel. The remaining seven genes have not been previously associated with resistance to paclitaxel in human cancer. Furthermore, we identified 815 (SKOV-3TR), 430 (OVCAR8TR) and 332 (MCF-7TR) transcripts that were more than twofold decreased relative to their expression in the corresponding parental cell line. Comparison of these transcripts identified three genes that were significantly underexpressed in all three drug-resistant cell lines, none of which have been previously associated with paclitaxel resistance.ConclusionsOur results confirm that the paclitaxel resistance phenotype is associated with a large number of transcriptional changes. In addition, acquired paclitaxel resistance was associated with distinct transcriptional changes in each of the cell lines studied, suggesting that paclitaxel resistance is a complex phenotype that can arise through multiple mechanisms.

GBP1 overexpression is associated with a paclitaxel resistance phenotype

In the search for novel genes involved in the paclitaxel resistance phenotype, prior studies of gene expression in paclitaxel-resistant cell lines and their paired drug-sensitive parental lines using high-density Affymetrix GeneChip arrays identified guanylate-binding protein 1 (GBP1) gene as an overexpressed transcript. The GBP1 gene encodes a large GTPase that is induced by interferon gamma (IFN-γ) in a variety of eukaryotic cells. In this report we characterize GBP1 and demonstrate that GBP1 expression is consistently upregulated in 7 of 8 paclitaxel or doxorubicin-resistant human cancer cell lines as compared to its expression in the relevant drug-sensitive parental lines. Analysis of GBP1 expression using the Cancer Profiling Array showed that GBP1 is ubiquitously expressed with no significant difference in expression levels between normal and tumor tissue. Parallel analysis of the Cancer Cell Line Profiling Array determined that GBP1 expression in a majority of cell lines derived from human tumors of different tissue origin was induced to variable levels following exposure to multiple stress agents including paclitaxel and doxorubicin. Importantly, stable expression of a GBP1 transgene in the paclitaxel-sensitive ovarian cancer cell line OVCAR8 was sufficient to confer moderate paclitaxel resistance. Our data suggest that increased expression of the GBP1 gene may play an important role in the development of multi-drug resistance (MDR).

Purinergic P2Y14 receptor modulates stress-induced hematopoietic stem/progenitor cell senescence

Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16(INK4a) expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury.

Cytogenetic abnormalities in products of conception: a relationship revisited.

Objectives:  Cytogenetic evaluation of product of conception (POC) is essential to determine the cause of pregnancy loss and aid the prenatal diagnosis of subsequent pregnancies. The purpose of this study is twofold. (1) To profile cytogenetic abnormalities, their relationship with maternal and gestational age and analyze sex ratios in our case series of 2052 consecutive samples of POC referred to the Baystate Medical Center, Laboratory Genetics between January 1992 and January 1999. (2) To present a comprehensive review of such data published in the last 15 years, in order to study temporal differences in the above parameters and make this information readily available for cytogeneticists and genetic counselors.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.