The ploidy conveyor of mature hepatocytes as a source of genetic variation.

Mononucleated and binucleated polyploid hepatocytes (4n, 8n, 16n and higher) are found in all mammalian species, but the functional significance of this conserved phenomenon remains unknown. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion. This raised the possibility that somatic 'reductive mitoses' can also happen in normal hepatocytes. Here we show that multipolar mitotic spindles form frequently in mouse polyploid hepatocytes and can result in one-step ploidy reversal to generate offspring with halved chromosome content. Proliferating hepatocytes produce a highly diverse population of daughter cells with multiple numerical chromosome imbalances as well as uniparental origins. Our findings support a dynamic model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a phenomenon that we term the 'ploidy conveyor'. We propose that this mechanism evolved to generate genetic diversity and permits adaptation of hepatocytes to xenobiotic or nutritional injury.

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

Extreme heterogeneity in the molecular events leading to the establishment of chiasmata during meiosis i in human oocytes.

In humans, ~50% of conceptuses are chromosomally aneuploid as a consequence of errors in meiosis, and most of these aneuploid conceptuses result in spontaneous miscarriage. Of these aneuploidy events, 70% originate during maternal meiosis, with the majority proposed to arise as a direct result of defective crossing over during meiotic recombination in prophase I. By contrast, <1%-2% of mouse germ cells exhibit prophase I-related nondisjunction events. This disparity among mammalian species is surprising, given the conservation of genes and events that regulate meiotic progression. To understand the mechanisms that might be responsible for the high error rates seen in human females, we sought to further elucidate the regulation of meiotic prophase I at the molecular cytogenetic level. Given that these events occur during embryonic development in females, samples were obtained during a defined period of gestation (17-24 weeks). Here, we demonstrate that human oocytes enter meiotic prophase I and progress through early recombination events in a similar temporal framework to mice. However, at pachynema, when chromosomes are fully paired, we find significant heterogeneity in the localization of the MutL homologs, MLH1 and MLH3, among human oocyte populations. MLH1 and MLH3 have been shown to mark late-meiotic nodules that correlate well with--and are thought to give rise to--the sites of reciprocal recombination between homologous chromosomes, which suggests a possible 10-fold variation in the processing of nascent recombination events. If such variability persists through development and into adulthood, these data would suggest that as many as 30% of human oocytes are predisposed to aneuploidy as a result of prophase I defects in MutL homolog-related events.

Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration.

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in many human solid tumors, and the level of its expression is associated with tumor neovascularization, invasiveness, and metastasis and with shorter patient survival time. TP promotes endothelial cell (EC) migration in vitro and angiogenesis in vivo, and these have been linked to its enzymatic activity. The mechanism by which TP stimulates EC migration was investigated using human umbilical vein ECs (HUVECs). TP induced concentration-dependent HUVEC migration, which required a TP gradient and thymidine and which was abrogated by the TP inhibitor CIMU (5-chloro-6(1-imidazolylmethyl)uracil). The chemotactic actions of TP plus thymidine were duplicated by the TP metabolite, 2-deoxyribose-1-phosphate (dR-1-P), and 10-fold more potently by its subsequent metabolite, 2-deoxyribose (2dR). Migration induced by dR-1-P, but not 2dR, was blocked by an alkaline phosphatase inhibitor, suggesting that the actions of dR-1-P first required its conversion to 2dR. In the migration assay, [5'-3H]dThd was metabolized to dR-1-P (96%) and 2dR (3.8%), and a gradient of both metabolites was maintained between the lower and upper chambers over the entire 5-h assay. TP expression in human solid tumors occurs in both tumor epithelial cells and in tumor-associated macrophages. The migration assay was adapted to use TP-transfected carcinoma cells to stimulate HUVEC migration, and they were found to induce more migration than did control vector-transfected cells. Human monocyte cells U937 and THP1, which constitutively expressed high levels of TP, also strongly induced HUVEC migration in the coculture assay. CIMU inhibited tumor-cell and monocyte-induced migration. In contrast, a neutralizing antibody to TP had no effect on cell-stimulated HUVEC migration, even though it completely blocked the migration mediated by purified TP. Thus, the intracellular actions of TP were sufficient to stimulate HUVEC chemotaxis. In contrast to purified TP, when incubated with [5'-3H]-thymidine, cells expressing TP released up to 20-fold more 2dR into the medium than dR-1-P. These studies demonstrate that TP-expressing cells mediate EC migration via the intracellular metabolism of thymidine and subsequent extracellular release of 2dR, which forms a chemotactic gradient.