Spectrum of Bmp5 Mutations from Germline Mutagenesis Experiments in Mice

Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGFβ superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals.     

KSHV Latency Locus Cooperates with Myc to Drive Lymphoma in Mice

Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma and B-cell malignancies. Mechanisms of KSHV-induced oncogenesis remain elusive, however, in part due to lack of reliable in vivo models. Recently, we showed that transgenic mice expressing the KSHV latent genes, including all viral microRNAs, developed splenic B cell hyperplasia with 100% penetrance, but only a fraction converted to B cell lymphomas, suggesting that cooperative oncogenic events were missing. Myc was chosen as a possible candidate, because Myc is deregulated in many B cell lymphomas. We crossed KSHV latency locus transgenic (latency) mice to Cα Myc transgenic (Myc) mice. By itself these Myc transgenic mice develop lymphomas only rarely. In the double transgenic mice (Myc/latency) we observed plasmacytosis, severe extramedullary hematopoiesis in spleen and liver, and increased proliferation of splenocytes. Myc/latency mice developed frank lymphoma at a higher rate than single transgenic latency or Myc mice. These data indicate that the KSHV latency locus cooperates with the deregulated Myc pathways to further lymphoma progression.     

Germline BAP1 Mutations Predispose to Renal Cell Carcinomas

The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.     

Germline Mutations in MAP3K6 Are Associated with Familial Gastric Cancer

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.     

应用蛋白截短技术检测APC基因胚系突变

建立蛋白截短检测技术,分析家族性腺瘤样息肉病（FAP）发病相关基因APC基因的胚系突变,研究基因突变型与FAP疾病表型的关系。经临床诊断的22例FAP患者及43例散发性大肠癌患者,分别取外周静脉血和正常结肠粘膜组织,常规提取基因组DNA。应用蛋白截短检测技术,分段分析APC基因巨大的第15外显子,对检出截短蛋白的样本进行测序,以确定突变位点及突变性质。22例FAP患者中,5例患者存在APC基因第15外显子的胚系突变,均为碱基缺失造成的移码突变,导致截短蛋白的产生;43例散发性大肠癌患者中未检测到APC基因第15外显子的胚系突变。蛋白截短检测技术是一种高效的基因突变分析技术,适用于大片段基因（如APC基因第15外显子）截短型突变的检测,可作为FAP症状出现前的常规基因诊断技术的一项有效补充。     

Reduced Intestinal Tumorigenesis in APCmin Mice Lacking Melanin-Concentrating Hormone

Background

Melanin-concentrating hormone (MCH) is an evolutionary conserved hypothalamic neuropeptide that in mammals primarily regulates appetite and energy balance. We have recently identified a novel role for MCH in intestinal inflammation by demonstrating attenuated experimental colitis in MCH deficient mice or wild type mice treated with an anti-MCH antibody. Therefore, targeting MCH has been proposed for the treatment of inflammatory bowel disease. Given the link between chronic intestinal inflammation and colorectal cancer, in the present study we sought to investigate whether blocking MCH might have effects on intestinal tumorigenesis that are independent of inflammation.

Methodology

Tumor development was evaluated in MCH-deficient mice crossed to the APCmin mice which develop spontaneously intestinal adenomas. A different cohort of MCH−/− and MCH+/+ mice in the APCmin background was treated with dextran sodium sulphate (DSS) to induce inflammation-dependent colorectal tumors. In Caco2 human colorectal adenocarcinoma cells, the role of MCH on cell survival, proliferation and apoptosis was investigated.

Results

APCmin mice lacking MCH developed fewer, smaller and less dysplastic tumors in the intestine and colon which at the molecular level are characterized by attenuated activation of the wnt/beta-catenin signaling pathway and increased apoptotic indices. Form a mechanistic point of view, MCH increased the survival of colonic adenocarcinoma Caco2 cells via inhibiting apoptosis, consistent with the mouse studies.

Conclusion

In addition to modulating inflammation, MCH was found to promote intestinal tumorigenesis at least in part by inhibiting epithelial cell apoptosis. Thereby, blocking MCH as a therapeutic approach is expected to decrease the risk for colorectal cancer.     

Altered Prostanoid Signaling Contributes to Increased Skin Tumorigenesis in Tpl2 Knockout Mice

Squamous cell carcinoma is the second most common form of skin cancer with the incidence expected to double over the next 20 years. Inflammation is believed to be a critical component in skin cancer progression. Therefore, understanding genes involved in the regulation of inflammatory pathways is vital to the design of targeted therapies. Numerous studies show cyclooxygenases (COXs) play an essential role in inflammation-associated cancers. Tpl2 (MAP3K8) is a protein kinase in the MAP Kinase signal transduction cascade. Previous research using a two-stage skin carcinogenesis model revealed that Tpl2 ^−/− mice have significantly higher tumor incidence and inflammatory response than wild-type (WT) controls. The current study investigates whether cyclooxygenase-2 (COX-2) and COX-2- regulated prostaglandins and prostaglandin receptors drive the highly tumorigenic state of Tpl2^−/− mice by investigating the relationship between Tpl2 and COX-2. Keratinocytes from newborn WT or Tpl2 ^−/− mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for various times over 24 hours. Western analysis revealed significant differences in COX-2 and COX-2 dependent prostanoids and prostanoid receptors. Additionally, in vivo experiments confirmed that COX-2 and COX-2 downstream factors were elevated in TPA-treated Tpl2^−/− skin, as well as in papillomas from Tpl2 ^−/− mice. Use of the selective COX-2 inhibitor Celecoxib showed the increased tumorigenesis in the Tpl2^−/− mice to primarily be mediated through COX-2. These experiments illustrate COX-2 induction in the absence of Tpl2 may be responsible for the increased tumorigenesis found in Tpl2 ^−/− mice. Defining the relationship between Tpl2 and COX-2 may lead to new ways to downregulate COX-2 through the modulation of Tpl2.     

Germline Mutations of Tetranucleotide DNA Repeats in Families with Normal Children and Reproductive Pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortions. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a panel of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortions with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 × 10⁻³ versus 2.32 × 10⁻³ per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.__________Translated from Genetika, Vol. 41, No. 7, 2005, pp. 943–953.Original Russian Text Copyright © 2005 by Nikitina, Lebedev, Sukhanova, Nazarenko.     

NLRP3 Inflammasome Activation Enhances ADK Expression to Accelerate Epilepsy in Mice

Epilepsy (SE) is a common and serious neurological disease. NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome participates in the pathogenesis of SE, while its underlying mechanism is still unclear. Here, we attempted to explore the mechanism of action of NLRP3 inflammasome in SE. SE mouse model was constructed by administration of kainic acid (KA). Astrocytes were treated with KA to mimic SE cell model. MCC950 (NLRP3 inhibitor) and Z-YVAD-FMK (Caspase-1 inhibitor) were used to treat astrocytes to inhibit the activity of NLRP3 and Caspase-1. Nissl staining was performed to examine the morphology of neuron. Western blot, enzyme-linked immunosorbent assay and immunofluorescence staining were performed to assess protein expression. SE mouse model exhibited an increase of neuronal loss, and an up-regulation of Cleaved-Caspase-1, IL-1β and IL-18 in hippocampus. The levels of GFAP⁺ADK⁺ cells were significantly increased in SE mice. MCC950 or Z-YVAD-FMK abolished these impacts conferred by KA in SE mice. Moreover, KA treatment enhanced the expression of NLRP3, Cleaved-Caspase-1, IL-1β and IL-18 in astrocytes, which was rescued by knockdown of NLRP3 or Caspase-1. Additionally, CREB, p-CREB, REST were up-regulated, and SP1 was down-regulated in the KA-treated SE mice and KA-treated astrocytes. Inhibition of NLRP3 or Caspase-1 rescued these proteins expression in KA-treated astrocytes. CREB or REST silencing reduced adenosine kinase (ADK) expression, while SP1 knockdown enhanced ADK expression in KA-treated astrocytes. In conclusion, NLRP3 inflammasome activation enhances ADK expression to accelerate SE in mice through regulating CREB/REST/SP1 signaling pathway. Thus, inhibition of NLRP3 inflammasome may be a treatment for SE.

     

PDGF-BB Does Not Accelerate Healing in Diabetic Mice with Splinted Skin Wounds

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epeithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.     

Germline and Somatic Mutations in Archived Breast Cancer Specimens of Different Subtypes

Molecular Biology - Molecular profiling of tumors may provide promising options for personalized treatment. We have examined the spectrum of germline and somatic mutations in 23 breast cancers (BC)...     

Gene Conversion between Unlinked Sequences in the Germline of Mice

Gene conversion between homologous sequences on non-homologous chromosomes (ectopic gene conversion) is remarkably frequent in fungi. It is thought to be a consequence of genome-wide homology scanning required to form synapses between homologous chromosomes. This activity provides a mechanism for concerted evolution of dispersed genes. Technical obstacles associated with mammalian systems have hitherto precluded investigations into ectopic gene conversion in the mammals. Here, we describe a binary transgenic mouse system to detect ectopic gene conversion in mice. Conversion events are visualized by histochemical staining of spermatids, and corroborated by polymerase chain reaction amplification of transgenes in spermatozoa. The results show that conversion between unlinked, hemizygous lacZ transgenes is frequent in the male germline, ranging from 0.1 to 0.7% of spermatids. Genomic location may affect the susceptibility to recombination, since the frequency varied between lines. The results suggest that homologous genes can undergo concerted evolution despite being genomically dispersed. However, mechanisms may exist to modulate this activity, enabling the divergence of duplicated genes.     

Abnormality in Wnt Signaling is Causatively Associated with Oxidative Stress-Induced Intestinal Tumorigenesis in MUTYH-Null Mice

MUTYH is a DNA glycosylase that excises adenine paired with 8-oxoguanine to prevent mutagenesis in mammals. Biallelic germline mutations of MUTYH have been found in patients predisposed to a recessive form of familial adenomatous polyposis (MAP: MUTYH-associated polyposis). We previously reported that Mutyh-deficient mice showed a high susceptibility to spontaneous and oxidative stress-induced intestinal adenoma/carcinoma. Here, we performed mutation analysis of the tumor-associated genes including Apc, Ctnnb1, Kras and Trp53 in the intestinal tumors of Mutyh-deficient mice. In the 62 tumors, we identified 25 mutations in Apc of 18 tumors and 36 mutations in Ctnnb1 of 36 tumors. Altogether, 54 out of the 62 tumors (87.1%) had a mutation in either Apc or Ctnnb1; no tumor displayed mutations simultaneously in the both genes. Similar to MAP, 60 out of 61 mutations (98.3%) were identified as G:C to T:A transversions of which 85% occurred at either AGAA or TGAA sequences. Immunohistochemical analyses revealed the accumulation of β-catenin in the nuclei of tumors. No mutation was found in either Kras or Trp53 in the tumors. These results indicate that the uncontrolled activation of Wnt signaling pathway is causatively associated with oxidative stress-induced intestinal tumorigenesis in the Mutyh-deficient mice.     

Developmental and Lactational Exposure to Dieldrin Alters Mammary Tumorigenesis in Her2/neu Transgenic Mice

Breast cancer is the most common cancer in Western women and while its precise etiology is unknown, environmental factors are thought to play a role. The organochlorine pesticide dieldrin is a persistent environmental toxicant thought to increase the risk of breast cancer and reduce survival in the human population. The objective of this study was to define the effect of developmental exposure to environmentally relevant concentrations of dieldrin, on mammary tumor development in the offspring. Sexually mature FVB-MMTV/neu female mice were treated with vehicle (corn oil), or dieldrin (0.45, 2.25, and 4.5 µg/g body weight) daily by gavage for 5 days prior to mating and then once weekly throughout gestation and lactation until weaning. Dieldrin concentrations were selected to produce serum levels representative of human background body burdens, occupational exposure, and overt toxicity. Treatment had no effect on litter size, birth weight or the number of pups surviving to weaning. The highest dose of dieldrin significantly increased the total tumor burden and the volume and number of tumors found in the thoracic mammary glands. Increased mRNA and protein expression of the neurotrophin BDNF and its receptor TrkB was increased in tumors from the offspring of dieldrin treated dams. This study indicates that developmental exposure to the environmental contaminant dieldrin causes increased tumor burden in genetically predisposed mice. Dieldrin exposure also altered the expression of BNDF and TrkB, novel modulators of cancer pathogenesis.     

High Frequency of Germline TP53 Mutations in a Prospective Adult-Onset Sarcoma Cohort

Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10⁻³), and earlier cancer onset (33 vs 48 years, P = 1.19×10⁻³). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.     

Germline and Somatic Mutations of Genes Involved in Tumor Formation in Sporadic Renal Angiomyolipoma

Russian Journal of Genetics - Angiomyolipoma (AML) is one of the most frequent and, at the same time, AML molecular genetics is one of the least studied among benign tumors. We performed deep...