ABSTRACT
We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 degrees C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.
Subject(s)
Cell Cycle Proteins/metabolism , Molecular Mimicry , Protein Transport , Animals , Antennapedia Homeodomain Protein/chemistry , Biphenyl Compounds , Cell Line, Tumor , Cells, Cultured , Clathrin/metabolism , Coated Pits, Cell-Membrane/chemistry , Coloring Agents/chemistry , Coloring Agents/metabolism , Endocytosis , Geminin , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Mice , NIH 3T3 Cells , Peptides/chemistry , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Peripheral B-cell lymphomas representing 90% of lymphoid neoplasms are divided into low- and high-growth fraction lymphomas. Here we investigate regulation of DNA replication licensing during B-cell lymphomagenesis. Combined analysis of origin licensing factors Mcm2 and geminin with the proliferation marker Ki67 in SLL/CLL, MCL, DLBCL and Burkitt lymphoma reveals for the first time the precise cell cycle state of these entities. Given that tight Mcm2 downregulation defines the quiescent state (G0) and that both high- and low-growth fraction lymphomas express Mcm2, the data demonstrate that neoplastic lymphocytes of SLL/CLL and MCL reside in an "in-cycle" G1 state and not in G0 as previously thought. Absence of the S/G2/M phase marker geminin in SLL/CLL and MCL further indicates failure of cell cycle progression in these tumours. In contrast, the high-growth fraction lymphomas DLBCL and Burkitt lymphoma exhibit differential expression of geminin, with the geminin/Ki67 ratio increasing for more aggressive neoplasms in keeping with a shortened G1 phase and thus representing an important discriminator for differential diagnosis. These data provide new insights into abrogation of cell cycle control during B cell lymphomagenesis and suggest that combined analysis of origin licensing factors may contribute to improved treatment decisions and prognosis in haematopoietic malignancies.
Subject(s)
DNA Replication , DNA, Neoplasm/genetics , Lymphoma, B-Cell/pathology , Biomarkers, Tumor/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Transformation, Neoplastic/pathology , Diagnosis, Differential , Flow Cytometry/methods , Geminin , Humans , Ki-67 Antigen/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphoma, B-Cell/diagnosis , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , Minichromosome Maintenance Complex Component 2 , Mitosis , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Tumor Cells, CulturedABSTRACT
The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.
Subject(s)
DNA Replication , Alternative Splicing , Animals , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Colon/metabolism , DNA/metabolism , Female , Flow Cytometry , Geminin , HL-60 Cells , HeLa Cells , Humans , Immunoblotting , Immunohistochemistry , Immunoprecipitation , Ki-67 Antigen/biosynthesis , Male , Meiosis , Minichromosome Maintenance Complex Component 2 , Mitosis , Nuclear Proteins/metabolism , Oocytes/metabolism , Prophase , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae Proteins/metabolism , Spermatogenesis , Testis/metabolism , Time Factors , Xenopus ProteinsABSTRACT
A method is describedforproducing sizable quantities of synchronously dividing, minimally disturbed mammalian cells. Cultures were grown immobilized on surfaces such that cell division within the population resulted in the continuous release of synchronous newborn cells. As judged by the quality and duration of synchronous growth, cell size distributions, and DNA compositions, newborn mouse L1210 cells grew with a very high level of synchrony without overt evidence of growth disturbances. The technology should be applicable to a variety of hematopoietic cells, as evidenced by similar results with human MOLT-4 and U937 cell lines.
