AIM: To assess mitotic stability of the fragile X full mutations and its relationship with DNA methylation.METHODS: The length change of the expanded CGG repeats was examined and correlated it with the methylation status in the DNA samples isolated from the fibroblasts derived from a fragile X female fetus and a fragile X male adult,respectively.RESULTS:A dramatic instability of the expanded CGG repeats in the female fetal fibroblasts was observed.Southern blot analysis revealed that the 6.9-kb major expanded band detected in passage 2 was completely replaced by a 7.7-kb band after passage 30.Fibroblast clones derived from the passage 3 displayed an unstable expansion of the CGG repeat during clonal proliferation,while methylation status of the CGG repeat region was maintained. In contrast,in fragile X male fibroblasts the expanded CGG repeats were stable during clonal proliferation. CONCLUSION:The mitotic instability of expanded CGG repeat is not always restricted in early development window as proposed previously and other elements rather than DNA methylation could affect the stability of the expanded CGG repeats in fragile X female fetal fibroblast cells.

PML bodies play an important role in multiple pathways of growth control,such as transformation suppression, apoptosis,and Ras-induced senescence. However,the molecular and biological bases for these physiological phenomena are not well understood. The findings that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery have suggested that PML bodies are transcription-permissible domains.To investigate the role of PML bodies in regulation of cell transformation and apoptosis-related gene transcription,we employed the immuno-FISH method to examine the relationship between PML bodies and the TP53 and BCL2 gene loci.PML bodies were found to localize specifically with the TP53 locus in about 50% of Jurkat interphase nuclei,but never in proximity with the BCL2 locus.We speculate that PML bodies may interact directly with the TP53 DNA sequence to regulate TP53 gene expression.

Translocation of the BCL2 gene from chromosome 18 to chromosome 14 results in constitutive expression of the gene.We have recently demonstrated that the major breakpoint region (mbr) of BCL2, which is implicated in 70% of t(14;18) translocations present in human follicular lymphoma, is a matrix attachment region.Since these regions are implicated in control of both transcription and replication,we wished to determine whether BCL2 translocation was also accompanied by changes in replication timing of the translocated allele.Using both fluorescence in situ hybridization and allele-specific PCR,we have demonstrated that the translocated allele replicates at the G1/S boundary,while the wildtype allele continues to replicate as usual in mid-S phase. These differences are accompanied by allele-specific changes in BCL2 expression.Since the net structural effect of t(14;18) translocations within the mbr is to disrupt the BCL2 MAR and replace it with the IGH MARs located just downstream of each breakpoint,we conclude that MAR exchange is a significant,selectable outcome of these translocations.We propose that subsequent changes of replication and transcriptional patterns for the translocated BCL2 allele result from this exchange and represent important early steps in lymphomagenesis.

In addition to inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9 activity,recent studies suggest that tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell lines.To address this question in pancreatic islets and β-cells,we treated rat pancreatic islets and INS-1 cells with a high-dose combination of the cytokines interleukin (IL)-1β,tumor necrosis factor-α, and interferon-g with or without the addition of TIMP-1 and TIMP-2 protein.Using flow cytometry,we quantitated DNA fragmentation to assess cellular apoptosis and confirmed these observations with DNA laddering experiments.Next,we transfected the mouse TIMP-1 gene into INS-1 cells and performed Western immunoblotting to demonstrate expression of TIMP-1 protein.We treated TIMP-1–expressing INS-1β cells with high-dose cytokines and again used flow cytometry to assess DNA fragmentation.We also evaluated the effect of TIMP-1 on IL-1β-induced inhibition of glucose-stimulated insulin secretion (GSIS) in freshly isolated rat pancreatic islets.Finally,we evaluated the effect of TIMP-1 on inducible nitric oxide synthase (iNOS) gene expression and nuclear factor (NF)-κB activity in INS-1 cells stimulated with high-dose cytokines.TIMP-1 but not TIMP-2 prevented cytokine-induced apoptosis and cytokine-mediated inhibition of GSIS in rat islets and β-cells.TIMP-1 mediated these effects by inhibiting cytokine activation of NF-κB,but it did not affect nitric oxide production or iNOS gene expression.Therefore,TIMP-1 may be an ideal gene to prevent cytokine-mediated β-cell destruction and dysfunction in models of type 1 diabetes and islet trans-plantation rejection.

Cyclooxygenase-2 (COX-2) gene and 12-lipoxygenase(12-1O) gene are preferentially expressed over other types of cyclooxygenase and lipoxygenase in pancreatic β-cells.Inhibition of either COX-2 or 12-LO can prevent cytokine-induced pancreatic β-cell dysfunction as defined by inhibition of glucose-stimulated insulin secretion.As cellular stress induces both genes and their respective end products in pancreatic in β-cells,we evaluated the role of 12-hydroxyeicosatetraenoic acid (HETE) on COX-2 gene expression,protein expression, and prostaglandin E2 (PGE2) production. We demonstrate that 12-HETE significantly increases COX-2 gene expression and consequent product formation,whereas a closely related lipid,15HETE,does not.In addition,IL-1β-stimulated prostaglandin E2 production is completely inhibited by a preferential lipoxygenase inhibitor cinnaminyl-3,4-dihydroxy-α-cyanocinnamate. We then evaluated IL-1β-induced PGE2 production in islets purified from control C57BL/6 mice and 12-LO knockout mice lacking cytokine-induc-ible 12-HETE.IL-1β stimulated an 8-fold increase in PGE2 production in C57BL/6 islets but failed to stimulate PGE2 in 12-LO knockout islets. Addition of 12-HETE to 12-LO knockout islet cells produced a statistically significant rise in PGE2 production. Furthermore,12-HETE, but not 15-HETE, stimulated COX-2 promoter and activator protein-1 binding activity.These data demonstrate that 12-HETE mediates cytokine-induced COX-2 gene transcription and resultant PGE2 production inpancreatic β-cells.(Molecular Endocrinology 16: 2145-2154, 2002)