Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.

Conventional anticancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter antitumor drug activity. To address this limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (for example, myeloma, leukemia and solid tumors) stably expressing luciferase are cultured with nonmalignant accessory cells (for example, stromal cells) for selective quantification of tumor cell viability, in presence versus absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib resistance in leukemic cells. A stroma-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-kappaB, HIF-1alpha, myc, hTERT and IRF4; for biological aggressiveness; and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, shows more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anticancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stroma interactions.

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.

Medulloblastoma is the most common malignant pediatric brain tumor, and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution single-nucleotide polymorphism genotyping arrays and subsequent interphase fluorescence in situ hybridization on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs (miRNA) in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated miRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the sonic hedgehog (Shh) signaling pathway compared with other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNP), proposed cells of origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.

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Circular RNAs (circRNAs) are RNA transcripts that are widespread in the eukaryotic genome. Recent evidence indicates that circRNAs play important roles in tissue development, gene regulation, and carcinogenesis. However, whether circRNAs encode functional proteins remains elusive, although translation of several circRNAs was recently reported.

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CircRNA deep sequencing was performed by using 10 pathologically diagnosed glioblastoma samples and their paired adjacent normal brain tissues. Northern blotting, Sanger sequencing, antibody, and liquid chromatograph Tandem Mass Spectrometer were used to confirm the existence of circ-FBXW7 and its encoded protein in in two cell lines. Lentivirus-transfected stable U251 and U373 cells were used to assess the biological functions of the novel protein invitro and invivo (five mice per group). Clinical implications of circ-FBXW7 were assessed in 38 pathologically diagnosed glioblastoma samples and their paired periphery normal brain tissues by using quantitative polymerase chain reaction (two-sided log-rank test).

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Circ-FBXW7 is abundantly expressed in the normal human brain (reads per kilobase per million mapped reads [RPKM] = 9.31). The spanning junction open reading frame in circ-FBXW7 driven by internal ribosome entry site encodes a novel 21-kDa protein, which we termed FBXW7-185aa. Upregulation of FBXW7-185aa in cancer cells inhibited proliferation and cell cycle acceleration, while knockdown of FBXW7-185aa promoted malignant phenotypes invitro and invivo. FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28-induced c-Myc stabilization. Moreover, circ-FBXW7 and FBXW7-185aa levels were reduced in glioblastoma clinical samples compared with their paired tumor-adjacent tissues (P < .001). Circ-FBXW7 expression positively associated with glioblastoma patient overall survival (P = .03).

UNASSIGNED

Endogenous circRNA encodes a functional protein in human cells, and circ-FBXW7 and FBXW7-185aa have potential prognostic implications in brain cancer.

The INK4a/ARF locus encodes two physically linked tumor suppressor proteins, p16(INK4a) and ARF, which regulate the RB and p53 pathways, respectively. The unusual genomic relationship of the open reading frames of these proteins initially fueled speculation that only one of the two was the true tumor suppressor, and loss of the other merely coincidental in cancer. Recent human and mouse genetic data, however, have firmly established that both proteins possess significant in vivo tumor suppressor activity, although there appear to be species- and cell-type specific differences between the two. For example, ARF plays a clear role in preventing Myc-induced lymphomagenesis in mice, whereas the role for p16(INK4a) is human carcinomas is more firmly established. In this review, I discuss the evolutionary history of the locus, the relative importance of these tumor suppressor genes in human cancer, and recent information suggesting novel biochemical and physiologic functions of these proteins in vivo.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

We have identified and cloned herculin, a fourth mouse muscle regulatory gene. Comparison of its DNA and deduced amino acid sequences with those of the three known myogenic genes (MyoD, myogenin, and Myf-5) reveals scattered short spans with similarity to one or more of these genes and a long span with strong similarity to all three. This long span includes a sequence motif that is also present in proteins of the myc, achaete-scute, and immunoglobulin enhancer-binding families. The herculin gene is physically linked to the Myf-5 gene on the chromosome; only 8.5 kilobases separate their translational start sites. A putative 27-kDa protein is encoded by three exons contained within a 1.7-kilobase fragment of the herculin gene. When expressed under the control of the simian virus 40 early promoter, transfected herculin renders murine NIH 3T3 and C3H/10T1/2 fibroblasts myogenic. In doing so, it also activates expression of myogenin, MyoD, and endogenous herculin in NIH 3T3 recipients. In adult mice, herculin is expressed in skeletal muscle but is absent from smooth muscle, cardiac muscle, and all nonmuscle tissues assayed. Direct comparison of the four known myogenic regulators in adult muscle showed that herculin is expressed at a significantly higher level than is any of the others. This quantitative dominance suggests an important role in the establishment or maintenance of adult skeletal muscle.

Cancer cells frequently have disease-specific chromosome rearrangements. It is poorly understood why translocations between chromosomes recur at specific breakpoints in the genome. Here we provide evidence that higher-order spatial genome organization is a contributing factor in the formation of recurrent translocations. We show that MYC, BCL and immunoglobulin loci, which are recurrently translocated in various B-cell lymphomas, are preferentially positioned in close spatial proximity relative to each other in normal B cells. Loci in spatial proximity are non-randomly positioned towards the nuclear interior in normal B cells. This locus proximity is the consequence of higher-order genome structure rather than a property of individual genes. Our results suggest that the formation of specific translocations in human lymphomas, and perhaps other tissues, is determined in part by higher-order spatial organization of the genome.

We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. Here, we have investigated mechanisms regulating the stability of Myc. We show that Myc is destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc. We also show that Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. Our data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.

Curcumin (diferuloylmethane), an anti-inflammatory agent used in traditional medicine, has been shown to suppress cellular transformation, proliferation, invasion, angiogenesis, and metastasis through a mechanism not fully understood. Because several genes that mediate these processes are regulated by nuclear factor-kappaB (NF-kappaB), we have postulated that curcumin mediates its activity by modulating NF-kappaB activation. Indeed, our laboratory has shown previously that curcumin can suppress NF-kappaB activation induced by a variety of agents (J Biol Chem 270:24995-50000, 1995). In the present study, we investigated the mechanism by which curcumin manifests its effect on NF-kappaB and NF-kappaB-regulated gene expression. Screening of 20 different analogs of curcumin showed that curcumin was the most potent analog in suppressing the tumor necrosis factor (TNF)-induced NF-kappaB activation. Curcumin inhibited TNF-induced NF-kappaB-dependent reporter gene expression in a dose-dependent manner. Curcumin also suppressed NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)1, TNFR2, NF-kappaB-inducing kinase, IkappaB kinase complex (IKK), and the p65 subunit of NF-kappaB. Such TNF-induced NF-kappaB-regulated gene products involved in cellular proliferation [cyclooxygenase-2 (COX-2), cyclin D1, and c-myc], antiapoptosis [inhibitor of apoptosis protein (IAP)1, IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-x(L), Bfl-1/A1, TNF receptor-associated factor 1, and cellular Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein-like inhibitory protein], and metastasis (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1) were also down-regulated by curcumin. COX-2 promoter activity induced by TNF was abrogated by curcumin. We found that curcumin suppressed TNF-induced nuclear translocation of p65, which corresponded with the sequential suppression of IkappaBalpha kinase activity, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Curcumin also inhibited TNF-induced Akt activation and its association with IKK. Glutathione and dithiothreitol reversed the effect of curcumin on TNF-induced NF-kappaB activation. Overall, our results indicated that curcumin inhibits NF-kappaB activation and NF-kappaB-regulated gene expression through inhibition of IKK and Akt activation.

The transcription factor GATA-1 is expressed in early hematopoietic progenitors and specifically down-regulated in myelomonocytic cells during lineage determination. Our earlier observation that the differentiation of Myb-Ets-transformed chicken hematopoietic progenitors into myeloblasts likewise involves a GATA-1 down-regulation, whereas expression is maintained in erythroid, thrombocytic, and eosinophilic derivatives, prompted us to study the effect of forced GATA-1 expression in Myb-Ets-transformed myeloblasts. We found that the factor rapidly suppresses myelomonocytic markers and induces a reprogramming of myeloblasts into cells resembling either transformed eosinophils or thromboblasts. In addition, we observed a correlation between the level of GATA-1 expression and the phenotype of the cell, intermediate levels of the factor being expressed by eosinophils and high levels by thromboblasts, suggesting a dosage effect of the factor. GATA-1 can also induce the formation of erythroblasts when expressed in a myelomonocytic cell line transformed with a Myb-Ets mutant containing a lesion in Ets. These cells mature into erythrocytes following temperature-inactivation of the Ets protein. Finally, the factor can reprogram a v-Myc-transformed macrophage cell line into myeloblasts, eosinophils, and erythroblasts, showing that the effects of GATA-1 are not limited to Myb-Ets-transformed myeloblasts. Our results suggest that GATA-1 is a lineage-determining transcription factor in transformed hematopoietic cells, which not only activates lineage-specific genetic programs but also suppresses myelomonocytic differentiation. They also point to a high degree of plasticity of transformed hematopoietic cells.

The muroid rodents are the largest superfamily of mammals, containing nearly one third of all mammal species. We report on a phylogenetic study comprising 53 genera sequenced for four nuclear genes, GHR, BRCA1, RAG1, and c-myc, totaling up to 6400 nucleotides. Most relationships among the subfamilies are resolved. All four genes yield nearly identical phylogenies, differing only in five key regions, four of which may represent particularly rapid radiations. Support is very strong for a fundamental division of the mole rats of the subfamilies Spalacinae and Rhizomyinae from all other muroids. Among the other "core" muroids, a rapid radiation led to at least four distinct lineages: Asian Calomyscus, an African clade of at least four endemic subfamilies, including the diverse Nesomyinae of Madagascar, a hamster clade with maximum diversity in the New World, and an Old World clade including gerbils and the diverse Old World mice and rats (Murinae). The Deomyinae, recently removed from the Murinae, is well supported as the sister group to the gerbils (Gerbillinae). Four key regions appear to represent rapid radiations and, despite a large amount of sequence data, remain poorly resolved: the base of the "core" muroids, among the five cricetid (hamster) subfamilies, within a large clade of Sigmodontinae endemic to South America, and among major geographic lineages of Old World Murinae. Because of the detailed taxon sampling within the Murinae, we are able to refine the fossil calibration of a rate-smoothed molecular clock and apply this clock to date key events in muroid evolution. We calculate rate differences among the gene regions and relate those differences to relative contribution of each gene to the support for various nodes. The among-gene variance in support is greatest for the shortest branches. We present a revised classification for this largest but most unsettled mammalian superfamily.

Deregulated expression of the c-myc proto-oncogene can lead to apoptosis under certain physiological conditions. By introducing a conditionally active Myc allele into primary embryo fibroblasts null for p53, and into fibroblasts without endogenous p53 expression but ectopically expressing a temperature-sensitive p53 allele, we show that expression of wild-type p53 is required for susceptibility to Myc-mediated apoptosis. Although ectopic expression of wild-type p53 blocked cells in the G1 phase of the cell cycle, G1 arrest by isoleucine starvation, in a manner independent of p53, did not confer susceptibility to apoptosis. Thus, growth arrest per se is not sufficient to induce Myc-mediated apoptosis; instead, a property intrinsic to p53 is specifically required. Moreover, apoptosis did not require induction of p53 target proteins, including the cyclin-dependent kinase inhibitor p21waf1/cip1. Therefore, the role of p53 in apoptosis may be distinct from its role in cell cycle arrest.

DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

The effect of the ionic products of Bioglass 45S5 dissolution on the gene-expression profile of human osteoblasts was investigated by cDNA microarray analysis of 1,176 genes. Treatment with the ionic products of Bioglass 45S5 dissolution increased the levels of 60 transcripts twofold or more and reduced the levels of five transcripts to one-half or less than in control. Markedly up-regulated genes included RCL, a c-myc responsive growth related gene, cell cycle regulators such as G1/S specific cyclin D1, and apoptosis regulators including calpain and defender against cell death (DAD1). Other significantly up-regulated genes included the cell surface receptors CD44 and integrin beta1, and various extracellular matrix regulators including metalloproteinases-2 and -4 and their inhibitors TIMP-1 and TIMP-2. The identification of differentially expressed genes by cDNA microarray analysis has offered new insights into the mode of action of bioactive glasses and has proven to be an effective tool in evaluating their osteoproductive properties.

The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways--one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.

Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

During post-lactational mammary gland involution, the bulk of mammary epithelium dies and is reabsorbed. This massive cell death and tissue restructuring was found to be accompanied by a specific pattern of gene expression. Northern blot analysis showed that weaning resulted in a dramatic drop in ODC, a gene involved in synthesis of a component of milk, and the nearly simultaneous induction of SGP-2, a gene associated with apoptotic cell death. These changes were followed by decreases in expression of milk protein genes to basal levels and expression of genes associated with regulation of cell proliferation and differentiation, p53, c-myc and TGF-beta 1. Subsequently, additional genes implicated in stress response, tissue remodelling, and apoptotic cell death were transiently expressed, expression peaking at about 6 days post-weaning. A non-random degradation of DNA yielding the oligonucleosomal length fragmentation pattern typical of apoptotic cell death (Wyllie, 1980; Wyllie et al., 1980) was detected in association with morphological changes and gene expression. The correlations between: (a) changes in morphology, (b) pattern of gene expression and (c) changes in DNA integrity suggest that complementary programs for cell death and tissue remodelling direct post-lactational mammary gland involution.

Hypoxia promotes genetic instability by undefined mechanisms. The transcription factor HIF-1alpha is crucial for the cellular response to hypoxia and is frequently overexpressed in human cancers, resulting in the activation of genes essential for cell survival. Here, we demonstrate that HIF-1alpha is responsible for genetic instability at the nucleotide level by inhibiting MSH2 and MSH6, thereby decreasing levels of the MSH2-MSH6 complex, MutSalpha, which recognizes base mismatches. HIF-1alpha displaces the transcriptional activator Myc from Sp1 binding to repress MutSalpha expression in a p53-dependent manner; Sp1 serves as a molecular switch by recruiting HIF-1alpha to the gene promoter under hypoxia. Furthermore, in human sporadic colon cancers, HIF-1alpha overexpression is statistically associated with the loss of MSH2 expression, especially when p53 is immunochemically undetectable. These findings indicate that the regulation of DNA repair is an integral part of the hypoxic response, providing molecular insights into the mechanisms underlying hypoxia-induced genetic instability.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.