Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2,6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP r ) fibroblast variants were recovered at a median frequency of 12 ؋ 10 ؊5 from individual ears from progeny of crosses between mouse strains 129͞Sv and C3H͞HeJ. The frequency of DAP r variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development. Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP r variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT. Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo.Retinoblastoma is a prototype disease for understanding how loss of function of tumor suppressor genes (TSGs) leads to tumor formation. The so-called two-hit (two-mutational events) model explains elegantly the inheritance of genetic predisposition and development of retinal tumors (1). In familial cases, a preexisting RB1 germ-line mutation (the first hit) is inherited, predisposing the retinoblast cells to tumor development by requiring only a second mutational event (the second hit). In sporadic cases, somatic cells lack the predisposing mutation, and a retinoblast cell must acquire two separate RB1 mutations to progress to a tumor. In the two-hit model, the first hit, a rate-limiting step, renders a cell heterozygous or hemizygous at RB1. The second hit, which is frequently referred to as loss of heterozygosity (LOH), leads to the expression of the RB1 mutant phenotype (2). Because

sFlt1-e15a emerged as an alternate transcript of Flt1 late in evolution with the insertion of an AluSq sequence into the primate genome after the emergence of the simian infraorder about 40 million years ago. sFlt1-e15a is particularly abundant in human placenta and trophoblasts and is also highly expressed in nonhuman primate placenta. The expressed protein has a C-terminal polyserine tail and, like reference sequence sFlt1 (sFlt1-i13), is glycosylated and secreted. Consistent with a role in placental pathophysiology, hypoxia stimulates sFlt1-e15a expression in isolated cytotrophoblasts and a trophoblast cell line, and differentiation into syncytiotrophoblasts further enhances the effect of hypoxia. Placental levels of sFlt1-e15a and sFlt1-i13 transcripts are significantly elevated in patients with preeclampsia compared with normal pregnancies. We speculate that sFlt1-e15a may contribute to the pathophysiology of preeclampsia.

Introduction Elevated circulating soluble FLT1 (sFLT1) levels in preeclampsia may play a role in its development. Aspirin is recommended for prevention of preeclampsia. We hypothesized that aspirin may inhibit the production of sFlt1. Methods Placentas from women with and without preeclampsia were collected. Primary cytotrophoblasts (CTBs) were cultured from normal placentas and treated with aspirin, sc-560, a COX1 inhibitor or celecoxib, a COX-2 inhibitor. The expression of sFLT1, FLT1, COX1, COX2 was studied. The effect of aspirin on sFlt1 expression was also studied in HEK293 cells and in HTR-8/SVNeo cells. Results The expression of sFLT1 was increased in preeclamptic placentas compared to control placentas and the expression and release of sFLT1 increased in CTBs exposed to 2% O2 compared to controls. Aspirin at 3 and 12 mM concentration reduced the expression and release of sFLT1 in CTBs. Aspirin also inhibited sFlt1 expression from HTR-8/SVNeo and HEK293 cells. Sc-560, but not celecoxib, reduced sFLT1 expression and release from CTBs. Aspirin and sc-560 also reduced hypoxia-induced FLT1 mRNA expression and inhibited COX1 mRNA in CTBs. Discussion This study confirms that sFLT1 expression is increased in preeclamptic placentas and in CTBs exposed to hypoxia. Aspirin inhibits the production sFLT1 in CTBs and in HTR-8/SVNeo. Sc-560 recapitulated the effects of aspirin on sFLT1 expression and release in CTBs suggesting that the aspirin effect may be mediated via inhibition of COX1. The study increases our understanding of the mechanisms regulating sFlt1 expression and provides a plausible explanation for the effect of aspirin to prevent preeclampsia.

FLT1 and its soluble form (sFLT1) arise as alternate transcripts from the same gene and sFLT1 can antagonize the effect of vascular endothelial growth factor (VEGF) on its cognate receptors. We investigated the effect of VEGF and protein kinase C (PKC) activation on sFLT1 abundance. We demonstrated that VEGF stimulates sFLT1 and FLT1 mRNA and protein levels in vascular endothelial cells via VEGFR2 and PKC. Using an FLT1 expression vector with N and C-terminal epitope tags, we show that PKC activation increases the cleavage of FLT1 into an N-terminal extracellular fragment and a C-terminal intracellular fragment with the cleavage occurring adjacent to the transmembrane domain. The trafficking and glycosylation inhibitors brefeldin, monensin and tunicamycin substantially reduced cleavage and release of the N-terminal ectodomain of FLT1 and inhibited secretion of the isoforms of sFLT1. The shed FLT1 ectodomain can bind VEGF and PlGF and inhibit VEGF-induced vascular tube formation thus confirming that it is functionally equivalent to the alternately spliced and secreted sFLT1 isoforms.