Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene.

Inherited susceptibility to uterine leiomyomas and renal cell cancer

Herein we report the clinical, histopathological, and molecular features of a cancer syndrome with predisposition to uterine leiomyomas and papillary renal cell carcinoma. The studied kindred included 11 family members with uterine leiomyomas and two with uterine leiomyosarcoma. Seven individuals had a history of cutaneous nodules, two of which were confirmed to be cutaneous leiomyomatosis. The four kidney cancer cases occurred in young (33- to 48-year-old) females and displayed a unique natural history. All these kidney cancers displayed a distinct papillary histology and presented as unilateral solitary lesions that had metastasized at the time of diagnosis. Genetic-marker analysis mapped the predisposition gene to chromosome 1q. Losses of the normal chromosome 1q were observed in tumors that had occurred in the kindred, including a uterine leiomyoma. Moreover, the observed histological features were used as a tool to diagnose a second kindred displaying the phenotype. We have shown that predisposition to uterine leiomyomas and papillary renal cell cancer can be inherited dominantly through the hereditary leiomyomatosis and renal cell cancer (HLRCC) gene. The HLRCC gene maps to chromosome 1q and is likely to be a tumor suppressor. Clinical, histopathological, and molecular tools are now available for accurate detection and diagnosis of this cancer syndrome.

The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

p19ARF targets certain E2F species for degradation

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse γ-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G2 + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and γ-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.

Direct DNA binding by Brca1

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein–DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Oncogenic potential of the adenovirus E4orf6 protein.

The group C adenovirus E4orf6 protein has previously been shown to bind to the p53 cellular tumor suppressor protein and block its ability to activate transcription. Here we show that the E4orf6 protein blocks the induction of p53-mediated apoptosis when AT6 cells, which harbor a temperature-sensitive p53, are shifted to the permissive temperature. The E4orf6 protein does not, however, prevent the induction of apoptosis in p53-deficient H1299 cells by treatment with tumor necrosis factor alpha and cycloheximide. The E4orf6 protein also cooperates with the adenovirus E1A protein to transform primary baby rat kidney cells, and it cooperates with the adenovirus E1A plus E1B 19-kDa and E1B 55-kDa proteins to increase the number of baby rat kidney cell transformants and enhance the rate at which they arise. The level of p53 is substantially reduced in transformed cells expressing the E4orf6 protein in comparison to adenovirus transformants lacking it. The E4orf6 gene also accelerates tumor formation when transformed baby rat kidney cells are injected subcutaneously into the nude mouse, and it converts human 293 cells from nontumorigenic to tumorigenic in nude mice. In addition to the well-studied E1A and E1B oncogenes, group C adenoviruses harbor a third oncogene, E4orf6, which functions in some respects similarly to the E1B oncogene.

Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts.

Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity. Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts. This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1. H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation. In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts. Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function.