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1.
Stem Cells ; 23(8): 1059-65, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16002779

ABSTRACT

The ability of cells to export Hoechst 33342 can be used to identify a subpopulation of cells (side population [SP]) with characteristics of stem cells in many tissues. The ATP-binding cassette transporters Bcrp1 (Abcg2) and Mdr1a/1b (Abcb1a/1b) have been implicated as being responsible for this phenotype. To further explore the involvement of these transporters in the SP phenotype, we have generated Bcrp1/Mdr1a/1b triple knockout mice and studied the effect of their absence on the SP in bone marrow and mammary gland. Whereas in bone marrow Bcrp1 was almost exclusively responsible for the SP, both transporters contributed to the SP phenotype in the mammary gland, where their combined absence resulted in a nearly complete loss of SP. Interestingly, bone marrow of Mdr1a/1b-/- mice frequently displayed an elevated SP, which was reversible by the Bcrp1 inhibitor Ko143, suggesting that Bcrp1 can compensate for the loss of Mdr1a/1b in bone marrow.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/physiology , ATP-Binding Cassette Transporters/physiology , Bone Marrow Cells/cytology , Mammary Glands, Animal/cytology , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Benzimidazoles , Bone Marrow Cells/metabolism , Cell Survival , Epithelial Cells/metabolism , Female , Flow Cytometry , Gene Silencing , Hematopoietic Stem Cells/metabolism , Immunohistochemistry , Mammary Glands, Animal/metabolism , Mice , Mice, Knockout , Phenotype , ATP-Binding Cassette Sub-Family B Member 4
2.
Blood ; 105(11): 4484-91, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15692065

ABSTRACT

B-cell chronic lymphocytic leukemia (B-CLL) is a clinically variable disease where mutations in DNA damage response genes ATM or TP53 affect the response to standard therapeutic agents. The in vitro cytotoxicity of a novel cyclin-dependent kinase inhibitor, CYC202, was evaluated in 26 B-CLLs, 11 with mutations in either the ATM or TP53 genes, and compared with that induced by ionizing radiation and fludarabine. CYC202 induced apoptosis within 24 hours of treatment in all 26 analyzed tumor samples independently of ATM and TP53 gene status, whereas 6 of 26 B-CLLs, mostly ATM mutant, showed marked in vitro resistance to fludarabine-induced apoptosis. Compared with B-CLLs, normal T and B lymphocytes treated with CYC202 displayed reduced and delayed apoptosis. Using global gene expression profiling, we found that CYC202 caused a significant down-regulation of genes involved in regulation of transcription, translation, survival, and DNA repair. Furthermore, induction of apoptosis by CYC202 was preceded by inhibition of RNA polymerase II phosphorylation, leading to down-regulation of several prosurvival proteins. We conclude that CYC202 is a potent inducer of apoptosis in B-CLL regardless of the functional status of the p53 pathway, and may be considered as a therapeutic agent to improve the outcome of resistant B-CLL tumors.


Subject(s)
Apoptosis/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Down-Regulation/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Purines/pharmacology , Transcription, Genetic/drug effects , Aged , Aged, 80 and over , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Survival/drug effects , Cell Survival/genetics , DNA-Binding Proteins/genetics , Down-Regulation/genetics , Drug Evaluation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Middle Aged , Mutation , Protein Serine-Threonine Kinases/genetics , RNA Polymerase II/metabolism , Roscovitine , Transcription, Genetic/genetics , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics
3.
Blood ; 103(1): 291-300, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-12958068

ABSTRACT

The ATM/p53-dependent DNA damage response pathway plays an important role in the progression of lymphoid tumors. Inactivation of the ATM or TP53 gene is frequent in B-cell lymphocytic leukemia (B-CLL) and leads to aggressive disease. Although the ATM and p53 pathways overlap, they are not congruent, and it is unclear how the mechanism of tumor progression differs between ATM- and p53-deficient tumors. Using microarray analysis of ATM-mutant, TP53-mutant, and ATM/TP53 wild-type B-CLLs, we show that after exposure to DNA damage transcriptional responses are entirely dependent on ATM function. The p53 proapoptotic responses comprise only a part of ATM-regulated transcription; additionally, ATM regulates prosurvival responses independently of p53. Consequently, the greater severity of the TP53-mutant B-CLLs compared with ATM-mutant B-CLLs is consistent with the additive effect of defective apoptotic and elevated survival responses after DNA damage in these tumors. We also show that transcription expression profiles of ATM-deficient, TP53-deficient, and wild-type B-CLLs are indistinguishable before irradiation. Therefore, damage-induced transcriptional fingerprinting can be used to stratify tumors according to their biologic differences and simultaneously identify potential targets for treating refractory tumors.


Subject(s)
Apoptosis/genetics , Genes, p53 , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Serine-Threonine Kinases/genetics , Apoptosis/radiation effects , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Cell Survival/genetics , Cell Survival/radiation effects , DNA Damage , DNA-Binding Proteins , Gene Expression Profiling , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/classification , Mutation , Oligonucleotide Array Sequence Analysis , Transcriptional Activation/radiation effects , Tumor Suppressor Proteins
4.
Breast Cancer Res ; 5(1): R1-8, 2003.
Article in English | MEDLINE | ID: mdl-12559051

ABSTRACT

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.


Subject(s)
Adipose Tissue/cytology , Breast/cytology , Mammary Glands, Animal/cytology , Neoplasm Proteins , 3T3 Cells , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Benzimidazoles/metabolism , Cell Differentiation , Cell Division/drug effects , Cell Transplantation , Cells, Cultured , Clone Cells/cytology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Flow Cytometry , Humans , Keratin-14 , Keratins/biosynthesis , Mice , RNA/genetics , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Verapamil/pharmacology
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