Caroline G. Lee

Genome-Wide Reprogramming in the Mouse Germ Line Entails the Base Excision Repair Pathway

Erasing Markers Epigenetic reprogramming of the mammalian genome, which involves the removal and replacement of the various regulatory epigenetic marks such as DNA methylation, occurs during germ cell differentiation and during early zygotic development. This process is also critical during the experimental generation of stem cells, but the factors and pathways that control epigenetic reprogramming are not well understood. Hajkova et al. (p. 78) investigated the erasure of DNA methylation during germ cell differentiation and during early zygotic development in the developing mouse and found that factors involved in the base excision repair (BER) pathway, which helps repair damaged DNA, were involved. Furthermore, inhibitors of BER resulted in the retention of DNA methylation in the zygote. Removing epigenetic marks early in mammalian development involves a DNA damage repair pathway. Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.

Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target

Like other cancers, aberrant gene regulation features significantly in hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) were recently found to regulate gene expression at the post-transcriptional/translational levels. The expression profiles of 157 miRNAs were examined in 19 HCC patients, and 19 up-regulated and 3 down-regulated miRNAs were found to be associated with HCC. Putative gene targets of these 22 miRNAs were predicted in silico and were significantly enriched in 34 biological pathways, most of which are frequently dysregulated during carcinogenesis. Further characterization of microRNA-224 (miR-224), the most significantly up-regulated miRNA in HCC patients, revealed that miR-224 increases apoptotic cell death as well as proliferation and targets apoptosis inhibitor-5 (API-5) to inhibit API-5 transcript expression. Significantly, miR-224 expression was found to be inversely correlated with API-5 expression in HCC patients (p < 0.05). Hence, our findings define a true in vivo target of miR-224 and reaffirm the important role of miRNAs in the dysregulation of cellular processes that may ultimately lead to tumorigenesis.

Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations.

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

MicroRNA and cancer – focus on apoptosis

•  Introduction •  miR biogenesis •  miR and Cancer •  miR and Apoptosis ‐  The pro‐apoptotic miRs targeting the BCL2 family of genes ‐  The anti‐apoptotic miR‐21 targets PTEN and PDCD4 ‐  miR‐210 decreases proapoptotic signalling in a hypoxic environment ‐  Let‐7/miR‐98 family and possible co‐operation with miR‐21 ‐  miR‐17–92 cluster highlights the complexity of miR regulatory networks ‐  miR‐224, the double‐edged sword ‐  Other miRs implicated in apoptosis •  Conclusion

Overexpression of Interleukin-13 Induces Minimal-Change–Like Nephropathy in Rats

IL-13 has been implicated in the pathogenesis of minimal-change nephrotic syndrome. This study aimed to investigate the role of IL-13 on the development of proteinuria and expression of podocyte-related genes that are associated with nephrotic syndrome. IL-13 was overexpressed in Wistar rats through transfection of a mammalian expression vector cloned with the rat IL-13 gene, into the quadriceps by in vivo electroporation. Serum IL-13, albumin, cholesterol, and creatinine and urine albumin were measured serially. Kidneys were harvested after day 70 for histology and electron microscopy. Glomerular gene expression of nephrin, podocin, dystroglycan, B7-1, and IL-13 receptor subunits were examined using real-time PCR with hybridization probes and expressed as an index against beta-actin. Protein expression of these molecules was determined by immunofluorescence staining. The IL-13-transfected rats (n = 41) showed significant albuminuria, hypoalbuminemia, and hypercholesterolemia when compared with control rats (n = 17). No significant histologic changes were seen in glomeruli of IL-13-transfected rats. However, electron microscopy showed up to 80% of podocyte foot process fusion. Glomerular gene expression was significantly upregulated for B7-1, IL-4Ralpha, and IL-13Ralpha2 but downregulated for nephrin, podocin, and dystroglycan. Immunofluorescence staining intensity was reduced for nephrin, podocin, and dystroglycan but increased for B7-1 and IL-4Ralpha in IL-13-transfected rats compared with controls. In conclusion, these results suggest that IL-13 overexpression in the rat could lead to podocyte injury with downregulation of nephrin, podocin, and dystroglycan and a concurrent upregulation of B7-1 in the glomeruli, inducing a minimal change-like nephropathy that is characterized by increased proteinuria, hypoalbuminemia, hypercholesterolemia, and fusion of podocyte foot processes.

Hepatitis B virus X gene and hepatocarcinogenesis

Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor in hepatocellular carcinoma (HCC), which is one of the most common cancers worldwide. The pathogenesis of HBV-mediated hepatocarcinogenesis is, however, incompletely understood. Evidence suggests that the HBV X protein (HBx) plays a crucial role in HCC development. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progression, apoptosis, protein degradation pathways, and genetic stability through interaction with host factors. This review describes the current state of knowledge of the molecular pathogenesis of HBV-induced HCC, with a focus on the role of HBx in hepatocarcinogenesis.

Normal Germ Line Establishment in Mice Carrying a Deletion of the Ifitm/Fragilis Gene Family Cluster

ABSTRACT The family of interferon-inducible transmembrane proteins (Ifitm) consists of five highly sequence-related cell surface proteins, which are implicated in diverse cellular processes. Ifitm genes are conserved, widely expressed, and characteristically found in genomic clusters, such as the 67-kb Ifitm family locus on mouse chromosome 7. Recently, Ifitm1 and Ifitm3 have been suggested to mediate migration of early primordial germ cells (PGCs), a process that is little understood. To investigate Ifitm function during germ cell development, we used targeted chromosome engineering to generate mutants which either lack the entire Ifitm locus or carry a disrupted Ifitm3 gene only. Here we show that the mutations have no detectable effects on development of the germ line or on the generation of live young. Hence, contrary to previous reports, Ifitm genes are not essential for PGC migration. The Ifitm family is a striking example of a conserved gene cluster which appears to be functionally redundant during development.

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter

3The MDR1 multidrug transporter P‐gp (P‐glycoprotein) is an efflux pump that extrudes diverse hydrophobic drugs and peptides from cells. Since the entry of HIV‐1 into cells involves an initial interaction of the viral gp41 hydrophobic peptide with the plasma membrane, a potential effect of P‐gp on HIV‐1 infectivity was explored. Virus production was greatly decreased when P‐gp was overexpressed at the surface of a continuous CD4+ human T‐leukemic cell line (12D7) infected with HIV‐1 NL4–3, a T‐tropic molecular clone of HIV‐1. P‐gp overexpression did not significantly alter the surface expression or distribution of either the HIV‐1 receptor CD4 or the coreceptor CXCR4. Reduction of HIV‐1 infectivity in P‐gp‐expressing cells occurred both during the fusion of viral and plasma membranes and at subsequent step(s) in the HIV‐1 life cycle.—Lee, C. G. L., Ramachandra, M., Jeang, K.‐T., Martin, M. A., Pastan, I., Gottesman, M. M. Effect of ABC transporters on HIV‐1 infection: inhibition of virus production by the MDR1 transporter. FASEB J. 14, 516–522 (2000)

MDR1, the blood–brain barrier transporter, is associated with Parkinson’s disease in ethnic Chinese

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease. It is characterised by bradykinesia, rigidity, resting tremor, and postural instability.1 It is a genetically heterogeneous disorder. Pathogenic mutations in several genes—including α-synuclein , Parkin , UCH-L1 (ubiquitin-C terminal hydrolase-L1) and DJ -1—have previously been identified in rare monogenic forms of this disease showing autosomal dominant, autosomal recessive, or maternal transmission, with or without genetic anticipation.2,3 The more common, sporadic form of Parkinson’s disease appears to result from an interaction between genetic and environmental factors.4 Polymorphisms in several genes, including those implicated in familial forms of the disease such as α-synuclein 5 and Parkin ,6,7 are also reported to be associated with the sporadic form.8 Genetic susceptibility to sporadic Parkinson’s disease was also found to be modulated by genes involved in xenobiotic management. A meta-analysis of 84 association studies of 14 genes showed that polymorphisms in four genes are significantly associated with the disease.9 These genes are either responsible for xenobiotic metabolism, such as NAT 210,11 and GST T1,12 or may interact with environmental agents, such as monoamine oxidase ( MAO B).13 Poor metaboliser alleles of the cytochrome P450 xenobiotic metabolism enzyme, CYP2D6, may also be associated with increased risk of Parkinson’s disease.14–20 Furthermore, there may be sex effects in the association of CYP 2D6 mutant alleles with Parkinson’s disease.21 These genetic association studies corroborate epidemiological studies, which have long suggested that Parkinson’s disease is associated with exposure to certain environmental xenobiotics. Although most of the specific agents remain to be identified, rural living, well water consumption, industrialisation, and herbicide/pesticide exposure have been implicated as potential risk factors.1,22,23 Another category of genes that may influence susceptibility to Parkinson’s disease is the …