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Biomedical subjects

Michael F Clarke

Publications and source records attributed to Michael F Clarke.

27 records · Page 2Linked to original sources
PubMed2003

In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.

Benzimidazoles↗
PubMed2003

Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells.

A central issue in stem cell biology is to understand the mechanisms that regulate the self-renewal of haematopoietic stem cells (HSCs), which are required for haematopoiesis to persist for the lifetime of the animal. We found that adult and fetal mouse and adult human HSCs express the proto-oncogene Bmi-1. The number of HSCs in the fetal liver of Bmi-1-/- mice was normal. In postnatal Bmi-1-/- mice, the number of HSCs was markedly reduced. Transplanted fetal liver and bone marrow cells obtained from Bmi-1-/- mice were able to contribute only transiently to haematopoiesis. There was no detectable self-renewal of adult HSCs, indicating a cell autonomous defect in Bmi-1-/- mice. A gene expression analysis revealed that the expression of stem cell associated genes, cell survival genes, transcription factors, and genes modulating proliferation including p16Ink4a and p19Arf was altered in bone marrow cells of the Bmi-1-/- mice. Expression of p16Ink4a and p19Arf in normal HSCs resulted in proliferative arrest and p53-dependent cell death, respectively. Our results indicate that Bmi-1 is essential for the generation of self-renewing adult HSCs.

Animals↗
PubMed2003

Prospective identification of tumorigenic breast cancer cells.

Breast cancer is the most common malignancy in United States women, accounting for >40,000 deaths each year. These breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Using a model in which human breast cancer cells were grown in immunocompromised mice, we found that only a minority of breast cancer cells had the ability to form new tumors. We were able to distinguish the tumorigenic (tumor initiating) from the nontumorigenic cancer cells based on cell surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44(+)CD24(-/low)Lineage(-) in eight of nine patients. As few as 100 cells with this phenotype were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. The tumorigenic subpopulation could be serially passaged: each time cells within this population generated new tumors containing additional CD44(+)CD24(-/low)Lineage(-) tumorigenic cells as well as the phenotypically diverse mixed populations of nontumorigenic cells present in the initial tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the elucidation of pathways that regulate their growth and survival. Furthermore, because these cells drive tumor development, strategies designed to target this population may lead to more effective therapies.

Animals↗
PubMed2003

Stem cells in normal breast development and breast cancer.

The main focus of this review is the role of mammary stem cells in normal breast development and carcinogenesis. We have developed a new in vitro culture system that permits, for the first time, the propagation of mammary stem and progenitor cells in an undifferentiated state, which should facilitate the elucidation of pathways that regulate normal mammary stem-cell self-renewal and differentiation. Furthermore, we propose a model in which transformation of stem cells, or early progenitor cells, results in carcinogenesis. A key event in this process is the deregulation of normal self-renewal in these cells. Transformed mammary stem or progenitor cells undergo aberrant differentiation processes that result in generation of the phenotypic heterogeneity found in human and rodent breast cancers. This phenotypic diversity is driven by a small subset of mammary tumour stem cells. We will discuss the important implications of this mammary tumour stem-cell model.

Animals↗
PubMed2002

New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication.

Oncolytic adenoviruses with restricted replication can be produced if the expression of crucial transcription units of the virus is controlled by tissue- or tumor-specific promoters. Here we describe a method for the rapid incorporation of exogenous promoters into the E1A and E4 regions of the human adenovirus type 5 genome. Using this system, we have generated AdEHT2 and AdEHE2F, two conditionally replicative adenoviruses for the treatment of breast cancer. The expression of the E1A gene in both viruses is controlled by a minimal dual-specificity promoter that responds to estrogens and hypoxia. The tight regulation of E1A expression correlated with the ability of these viruses to replicate and kill human cancer cells that express estrogen receptors, or are maintained under hypoxic conditions. The telomerase reverse transcriptase (TERT) promoter and the E2F-1 promoter are preferentially activated in cancer cells. They were introduced into the E4 region of AdEHT2 and AdEHE2F, respectively. The telomerase core promoter failed to block the replication of the virus in telomerase-negative cells. In contrast, AdEHE2F was attenuated in nontransformed quiescent cells growing under normoxic conditions, suggesting that an intact pRB pathway with low levels of E2F transcription factors acts as a negative modulator for the virus. These data indicate that the simultaneous regulation of E1A and E4 viral transcription units by the appropriate combination of promoters can increase the tumor selectivity of oncolytic adenoviruses.

Adenocarcinoma↗
PubMed2002

Differential gene expression profiling of adult murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

Animals↗
PubMed2002

A genetic determinant that specifically regulates the frequency of hematopoietic stem cells.

The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.

Animals↗
PubMed2002

SLUGging away at cell death.

Programmed cell death (PCD) plays an important role in normal and malignant hematopoieis. In this issue of Cancer Cell, Inoue et al. (2002) demonstrate that the CED-1 homolog, Slug, is a key regulator of apoptosis in the response of early hematopoietic progenitors to gamma radiation.

Animals↗