Background

Genome-wide association studies (GWAS) have identified many common polymorphisms associated with complex traits. However, these associated common variants explain only a small fraction of the phenotypic variances, leaving a substantial portion of genetic heritability unexplained. As a result, searches for "missing" heritability are drawing increasing attention, particularly for rare variant studies that often require a large sample size and, thus, extensive sequencing effort. Although the development of next generation sequencing (NGS) technologies has made it possible to sequence a large number of reads economically and efficiently, it is still often cost prohibitive to sequence thousands of individuals that are generally required for association studies. A more efficient and cost-effective design would involve pooling the genetic materials of multiple individuals together and then sequencing the pools, instead of the individuals. This pooled sequencing approach has improved the plausibility of association studies for rare variants, while, at the same time, posed a great challenge to the pooled sequencing data analysis, essentially because individual sample identity is lost, and NGS sequencing errors could be hard to distinguish from low frequency alleles.

Results

A unified approach for estimating minor allele frequency, SNP calling and association studies based on pooled sequencing data using an expectation maximization (EM) algorithm is developed in this paper. This approach makes it possible to study the effects of minor allele frequency, sequencing error rate, number of pools, number of individuals in each pool, and the sequencing depth on the estimation accuracy of minor allele frequencies. We show that the naive method of estimating minor allele frequencies by taking the fraction of observed minor alleles can be significantly biased, especially for rare variants. In contrast, our EM approach can give an unbiased estimate of the minor allele frequency under all scenarios studied in this paper. A SNP calling approach, EM-SNP, for pooled sequencing data based on the EM algorithm is then developed and compared with another recent SNP calling method, SNVer. We show that EM-SNP outperforms SNVer in terms of the fraction of db-SNPs among the called SNPs, as well as transition/transversion (Ti/Tv) ratio. Finally, the EM approach is used to study the association between variants and type I diabetes.

Conclusions

The EM-based approach for the analysis of pooled sequencing data can accurately estimate minor allele frequencies, call SNPs, and find associations between variants and complex traits. This approach is especially useful for studies involving rare variants.

Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

Background

Internal migrant workers are a large population in China. Current health related studies among this population mainly focused on infectious disease, maternal health and occupational diseases and injuries. However, very limited studies were paid attention to mental health of migrant workers though it is an important public health issue.

Aims

The current study aims to understand prevalence of depression symptoms and factors associated with depression among Chinese migrant workers using novel methods to develop a comprehensive sample.

Methods

Respondent-driven sampling (RDS) was employed to recruit the target population, who are required 1) not to hold a hukou indicative of living in central areas or near suburbs of Chengdu city; 2) to be 16 years or older; 3) not to be a student. The Center for Epidemiologic Depression Scale (CES-D) was used to measure depression symptoms of migrant workers. And then Structural Equation Model (SEM) was applied to explore factors associated with depression among Chinese migrant workers.

Results

Among 1,180 migrant workers, 23.7% of them had clinically relevant depression symptoms (CES-D score >= 16), and 12.8% were consistent with a clinical diagnosis of depression (CES-D score >= 21). Self-rated economic status, city adaptation status, and self-rated health had negative effects on depression. Social economic status (SES) affected depression, and was mediated by self-rated economic status and self-rated health. City adaptation status was affected by length of residence in the city, satisfaction with one’s job, and the social support that one could obtain while living in the city.

Conclusions

The findings indicated a higher prevalence of depression symptoms among migrant workers comparing to general population reported by previous studies, identified possible factors associated with depression symptoms, and also explored relationships between these factors. Our study provides a model to understand mental health of Chinese internal migrant workers and to generate important research questions for the future.

Genome-wide association studies have identified prostate cancer susceptibility alleles on chromosome 11q13. As part of the Cancer Genetic Markers of Susceptibility (CGEMS) Initiative, the region flanking the most significant marker, rs10896449, was fine mapped in 10 272 cases and 9123 controls of European origin (10 studies) using 120 common single nucleotide polymorphisms (SNPs) selected by a two-staged tagging strategy using HapMap SNPs. Single-locus analysis identified 18 SNPs below genome-wide significance (P< 10−8) with rs10896449 the most significant (P= 7.94 × 10−19). Multi-locus models that included significant SNPs sequentially identified a second association at rs12793759 [odds ratio (OR) = 1.14, P= 4.76 × 10−5, adjusted P= 0.004] that is independent of rs10896449 and remained significant after adjustment for multiple testing within the region. rs10896438, a proxy of previously reported rs12418451 (r2= 0.96), independent of both rs10896449 and rs12793759 was detected (OR = 1.07, P= 5.92 × 10−3, adjusted P= 0.054). Our observation of a recombination hotspot that separates rs10896438 from rs10896449 and rs12793759, and low linkage disequilibrium (rs10896449–rs12793759, r2= 0.17; rs10896449–rs10896438, r2= 0.10; rs12793759–rs10896438, r2= 0.12) corroborate our finding of three independent signals. By analysis of tagged SNPs across ∼123 kb using next generation sequencing of 63 controls of European origin, 1000 Genome and HapMap data, we observed multiple surrogates for the three independent signals marked by rs10896449 (n= 31), rs10896438 (n= 24) and rs12793759 (n= 8). Our results indicate that a complex architecture underlying the common variants contributing to prostate cancer risk at 11q13. We estimate that at least 63 common variants should be considered in future studies designed to investigate the biological basis of the multiple association signals.

Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission.

The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.

Background

Metabolic syndrome traits play an important role in the development of colorectal cancer. Adipokines, key metabolic syndrome cellular mediators, when abnormal, may induce carcinogenesis.

Methodology/Principal Findings

To investigate whether polymorphisms of important adipokines, adiponectin (ADIPOQ) and its receptors, either alone or in combination with environmental factors, are implicated in colorectal cancer, a two-stage case-control study was conducted. In the first stage, we evaluated 24 tag single nucleotide polymorphisms (tag SNPs) across ADIPOQ ligand and two ADIPOQ receptors (ADIPOR1 and ADIPOR2) among 470 cases and 458 controls. One SNP with promising association was then analyzed in stage 2 among 314 cases and 355 controls. In our study, ADIPOQ rs1063538 was consistently associated with increased colorectal cancer risk, with an odds ratio (OR) of 1.94 (95%CI: 1.48–2.54) for CC genotype compared with TT genotype. In two-factor gene-environment interaction analyses, rs1063538 presented significant interactions with smoking status, family history of cancer and alcohol use, with ORs of 4.52 (95%CI: 2.78–7.34), 3.18 (95%CI: 1.73–5.82) and 1.97 (95%CI: 1.27–3.04) for smokers, individuals with family history of cancer or drinkers with CC genotype compared with non-smokers, individuals without family history of cancer or non-drinkers with TT genotype, respectively. Multifactor gene-environment interactions analysis revealed significant interactions between ADIPOQ rs1063538, ADIPOR1 rs1539355, smoking status and BMI. Individuals carrying one, two and at least three risk factors presented 1.18–fold (95%CI:0.89–fold to 1.58–fold), 1.87–fold (95%CI: 1.38–fold to2.54–fold) and 4.39–fold (95%CI: 2.75–fold to 7.01–fold) increased colorectal cancer risk compared with those who without risk factor, respectively (P trend <0.0001).

Conclusions/Significance

Our results suggest that variants in ADIPOQ may contribute to increased colorectal cancer risk in Chinese and this contribution may be modified by environmental factors, such as smoking status, family history of cancer and BMI.

Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy.

A total of $275 million has been launched to The Cancer Genome Atlas Project for genomic mapping of more than 20 types of cancers. The major challenge is to develop high throughput and cost-effective techniques for human genome sequencing. We developed a targeted exome sequencing technology to routinely determine human exome sequence. As a proof-of-concept, we chose a unique patient, who underwent three high mortalities cancers, i.e., breast, gallbladder and lung cancers, to reveal the genetic cause of high-cancer-susceptibility. Total 24,545 SNPs were detected. 10,868 (44.27%) SNPs were within coding regions, and 1,077 (4.38%) located in the UTRs. 3367 genes were hit by 4480 non-sysnonymous mutations in CDS with truncation of 30 proteins; and 10 mutations occurred at the splice sites that would generate different protein isoforms. Substitutions or premature terminations occurred in 132 proteins encoded by cancer-associated genes. CARD8 was completely loss; ANAPC1 was pre-translationally terminated from the transcripts of one allele. On the Ras-MAPK pathway, 18 genes were homozygously mutated. 15 growth factors/cytokines and their receptors, 9 transcription factors, 6 proteins on WNT signaling pathway, and 16 cell surface and extracellular proteins may be dysfunctioned. Exome sequencing made it possible for individualized cancer therapy.

Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.

DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

Author Summary

Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.

The precise molecular mechanism underlying arsenic trioxide (As2O3)-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As2O3 can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As2O3 directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As2O3 responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As2O3-induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As2O3-induced cyto c release, which could be specifically inhibited by Bcl-XL. Third, As2O3 induced mitochondria membrane potential (ΔΨm) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As2O3 induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.

Purpose

To investigate delivery quality assurance (DQA) discrepancies observed for a subset of helical tomotherapy patients.

Methods and Materials

Six tomotherapy patient plans were selected for analysis. Three had passing DQA ion chamber (IC) measurements while three had measurements deviating from the expected dose by more than 3.0%. All plans utilized similar parameters, including: 2.5 cm field-width, 15 s gantry period, and pitch values ranging from 0.143–0.215. Preliminary analysis suggested discrepancies were associated with plans having predominantly small leaf open times (LOTs). To test this, patients with failing DQA measurements were replanned using an increased pitch of 0.287. New DQA plans were generated and IC measurements performed. Exit fluence data was also collected during DQA delivery for dose reconstruction purposes.

Results

Sinogram analysis showed increases in mean LOTs ranging from 29.8–83.1% for the increased pitch replans. IC measurements for these plans showed a reduction in dose discrepancies, bringing all measurements within ±3.0%. The replans were also more efficient to deliver, resulting in reduced treatment times. Dose reconstruction results were in excellent agreement with IC measurements, illustrating the impact of leaf-timing inaccuracies on plans having predominantly small LOTs.

Conclusions

The impact of leaf-timing inaccuracies on plans with small mean LOTs can be considerable. These inaccuracies result from deviations in MLC leaf latency from the linear approximation used by the treatment planning system and can be important for plans having a 15 s gantry period. The ability to reduce this effect while improving delivery efficiency by increasing the pitch is demonstrated.

Cytotoxic activities of jadomycin B and five new jadomycin derivatives against four cancer cell lines (HepG2, IM-9, IM-9/Bcl-2 and H460) were evaluated. Jadomycin S was most potent against HepG2, IM-9 and IM-9/Bcl-2 while jadomycin F was most potent against H460. Their potencies correlated with the degrees of apoptosis induced. Structure-activity-relationship analyses clearly demonstrate that the side chains of the oxazolone ring derived from the incorporated amino acids make a significant impact on biological activity. Therefore, jadomycin offers an ideal scaffold to manipulate structure and could be exploited to make many novel bioactive compounds with altered activities.

It is known that head and neck squamous cell carcinomas (HNSCC) originating from different anatomic locations can exhibit varying behavior that is not predictable by histopathology of the primary tumor. Using a microarray containing 27,323 cDNA clones, we generated sets of gene expression profiles for 36 HNSCC primary tumors (12 oral cavity, 12 oropharynx, and 12 larynx/hypopharynx). From these datasets, we ranked genes according to their ability to differentiate between patients whose disease progressed within a 24 month period (aggressive phenotype) and those that did not (non-aggressive phenotype) based on levels of gene expression. A merging of datasets from the three sites revealed that only a fraction of identified genes were shared between any two sites. This contrasted greatly with the significant overlap (approximately 50%) in down-regulated genes identified in tumor/normal comparisons using cases both from oropharynx and larynx/hypopharynx. From these data, we conclude that HNSCC tumors originating from different anatomic sites share consistent changes in gene expression when comparing primary tumors to normal adjacent mucosa; these common changes most likely reflect alterations required for tumor development. In contrast, once a tumor has developed, tumor-host interactions at the different anatomic sites are likely responsible for the site-specific signatures associated with aggressive versus non-aggressive disease. Predictions of outcome based on gene expression profiling are therefore heavily influenced by the anatomic site of the primary tumor.

Spatial-angular compounding is a new technique that enables the reduction of noise artifacts in ultrasound elastography. Under this method, compounded elastograms are obtained from a spatially weighted average of local strain estimated from radio frequency (rf) echo signals acquired at different insonification angles. In previous work, the acquisition of the rf signals was performed through the lateral translation of a phased-array transducer. Clinical applications of angular compounding would, however, require the utilization of beam steering on linear-array transducers to obtain angular data sets, which is more efficient than translating phased-array transducers. In this article, we investigate the performance of angular compounding for elastography by using beam steering on a linear-array transducer. Quantitative experimental results demonstrate that spatial angular compounding provides significant improvement in both the elastographic signal-to-noise ratio and the contrast-to-noise ratio. For the linear array transducer used in this study, the optimum angular increment is around 1.5°–3.75°, and the maximum angle that can be used in angular compounding should not exceed 10°.

A theoretical analysis of the correlation between radio-frequency (RF) echo signal data acquired from the same location but at different angles is presented. The accuracy of the theoretical results is verified with computer simulations. Refinements to previous analyses of the correlation of RF signals originating from the same spatial location at different angular positions are made. We extend the analysis to study correlation of RF signals coming from different spatial locations and eventually correlation of RF signal segments that intersect at the same spatial location. The theory predicts a faster decorrelation with a change in the insonification angle for longer RF echo signal segments. As the RF signal segment becomes shorter, the decorrelation rate with angle is slower and approaches the limit corresponding to the correlation of RF signals originating from the same spatial location. Theoretical results provide a clear understanding of angular compounding techniques used to improve the signal-to-noise ratio in ultrasonic parametric imaging and in elastography.

Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine proteases (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.

Apo2 Ligand or Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) is a member of the TNF gene superfamily that selectively induces apoptosis in tumor cells of diverse origins through engagement of death receptors. We have recently demonstrated that Type I interferons (IFN-α and β) induce apoptosis in multiple myeloma (MM) cell lines and in plasma cells from MM patients. Moreover, Apo2L selectively induces apoptosis of patient MM tumor cells while sparing non-malignant cells. Apo2L induction is one of the earliest events following IFN administration in these cells. IFNs activate Caspases and the mitochondrial-dependent apoptotic pathway mediated by Apo2L production. Cell death induced by IFNs and Apo2L can be blocked by a dominant-negative Apo2L receptor, DR5, and is regulated by members of the Bcl-2 family of proteins. This review is focused on the apoptotic signaling pathways regulated by Apo2L and Bcl-2-family proteins and summarizes what is known about their clinical role.

Background

Wnt/β-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/β-catenin pathway modification of the cellular apoptosis.

Methods

To identify potential genes regulated by Wnt/β-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of β-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR.

Results

Stably integrated inducible RNAi vector could effectively suppress β-catenin expression and the transcriptional activity of β-catenin/TCF. Meanwhile, depletion of β-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of β-catenin.

Conclusion

Coupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/β-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified β-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth.

It has been reported that interferons (IFNs) may have antitumor activity in multiple myeloma (MM). The mechanism for their effect on MM, however, remains elusive. This study shows that IFN-α and -β, but not -γ, induce apoptosis characterized by Annexin V positivity, nuclear fragmentation and condensation, and loss of clonogenicity in 3 MM cell lines (U266, RPMI-8266, and NCI-H929), and in plasma cells from 10 patients with MM. Apo2 ligand (Apo2L, also TRAIL) induction was one of the earliest events following IFN administration in U266 cells. Treatment of these cells with TRAIL, but not with Fas agonistic antibodies, induces apoptosis. Cell death induced by IFNs and Apo2L in U266 cells was partially blocked by a dominant-negative Apo2L receptor, DR5, demonstrating the functional significance of Apo2L induction. This study shows that IFNs activate caspases and the mitochondrial-dependent apoptotic pathway, possibly mediated by Apo2L production. Thus, IFN-α and -β induce cytochrome c release from mitochondria starting at 12 hours, with an amplified release seen at 48 hours. Moreover, Bid cleavage precedes the initial cytochrome c release, whereas the late, amplified cytochrome c release coincides with changes in levels of Bcl-2, Bcl-XL, and reduction of mitochondrial membrane potential. These results link the Apo2L induction and modulation of Bcl-2 family proteins to mitochondrial dysfunction. Furthermore, IFNs and Apo2L induce cell death of CD38+/CD45−/dim plasma cells, without significant effect on nonplasma blood cells, in a caspase and Bcl-2 cleavage-dependent manner. These results warrant further clinical studies with IFNs and Apo2L in MM.

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E276-395 (where cyclin E276-395 is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E1-275, cyclin E276-395 deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E276-395, but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.