ABHD6 suppression promotes anti-inflammatory polarization of adipose tissue macrophages via 2-monoacylglycerol/PPAR signaling in obese mice.

OBJECTIVE: Pro-inflammatory polarization of adipose tissue macrophages (ATMs) plays a critical role in the pathogenesis of obesity-associated chronic inflammation. However, little is known about the role of lipids in the regulation of ATMs polarity and inflammation in response to metabolic stress. Deletion of α/ß-hydrolase domain-containing 6 (ABHD6), a monoacylglycerol (MAG) hydrolase, has been shown to protect against diet-induced obesity and insulin resistance. METHODS: Here we investigated the immunometabolic role of macrophage ABHD6 in response to nutrient excess using whole-body ABHD6-KO mice and human and murine macrophage cell-lines treated with KT203, a selective and potent pharmacological ABHD6 inhibitor. RESULTS: KO mice on high-fat diet showed lower susceptibility to systemic diet-induced inflammation. Moreover, in the setting of overnutrition, stromal vascular cells from gonadal fat of KO vs. control mice contained lower number of M1 macrophages and exhibited enhanced levels of metabolically activated macrophages (MMe) and M2 markers, oxygen consumption, and interleukin-6 (IL-6) release. Likewise, under in vitro nutri-stress condition, inhibition of ABHD6 in MMe-polarized macrophages attenuated the expression and release of pro-inflammatory cytokines and M1 markers and induced the upregulation of lipid metabolism genes. ABHD6-inhibited MMe macrophages showed elevated levels of peroxisome proliferator-activated receptors (PPARs) and 2-MAG species. Notably, among different MAG species, only 2-MAG treatment led to increased levels of PPAR target genes in MMe macrophages. CONCLUSIONS: Collectively, our findings identify ABHD6 as a key component of pro-inflammatory macrophage activation in response to excess nutrition and implicate an endogenous macrophage lipolysis/ABHD6/2-MAG/PPARs cascade, as a lipid signaling and immunometabolic pathway, which favors the anti-inflammatory polarization of ATMs in obesity.

Age-dependent penetrance of different germline mutations in the BRCA1 gene.

AIMS: BRCA1 gene mutations have been extensively studied in relation to breast and ovarian cancer susceptibility. Various genotype-phenotype correlation attempts have yielded important data pertaining to the consequences of BRCA1 mutations. However, little is known about the effects of recurrent BRCA mutations on expressivity and the age of onset of cancer in a population. This study addresses whether different exon mutations have variable expressivity especially in relation to the age of onset of breast cancer. METHODS: Using a step-wise systematic approach, culminating in the sequencing of all BRCA1 and BRCA2 exons with the addition of multiplex ligation-dependent probe amplification, the relationship between disease phenotypes and gene mutations in 219 individuals and their family members was examined. RESULTS: It is shown that different BRCA1 gene mutations have distinct effects that influence the age of onset of breast or ovarian cancer. Mutations in exon 2 of the BRCA1 gene had significantly lower penetrance compared with mutations of exons 11, 13 and 20. The median age of affliction with breast cancer was 55 years for 185delAG in exon 2 (95% confidence interval (CI) 46.7 to 59.5), 47 years for the 4184delTCAA mutation in exon 11 (95% CI 39 to 55.4), and 41 years for exon 13 duplication (95% CI 32.9 to 49.7) of the BRCA1 gene. Moreover, 14 novel mutations in BRCA1 and BRCA2 genes in the Yorkshire/Humberside population were identified. CONCLUSIONS: The 185delAG mutation of the BRCA1 gene is a low penetrance mutation that is age dependent especially when compared with the exon 13 duplication mutation. The data have important ramifications on screening, genetic counselling and prophylactic treatment of BRCA1 gene mutation carriers.

Could nuclear matrix protein 22 (NMP22) play a role with urine cytology in screening for bladder cancer?--experience at Kuwait University.

OBJECTIVES: This prospective study was undertaken to evaluate nuclear matrix protein (NMP22) compared to urine cytology in the detection of bladder cancer and also to determine whether indexing suspicious cytology to NMP22 could enhance the clinical utility of cytology. METHODS: Cytological findings of voided urine collected prior to a cystoscopic biopsy were correlated with urine NMP22 assay in 46 patients attending the urology clinic in Mubarak Al-Kabeer Hospital. The patients were clinically categorized into newly diagnosed cases of transitional cell carcinoma (TCC), recurrent TCC, TCC in remission and controls. RESULTS: Using histological diagnosis as the gold standard the sensitivity and specificity of NMP22 were 78% and 43% respectively and of cases with malignant urine cytology were 30% and 87% respectively. If suspicious and malignant cytology were combined as positive results the sensitivity increased significantly to 87% while the specificity decreased but not significantly to 74%. Suspicious or malignant cytology enhanced by positive NMP22 gave a sensitivity of 70% and specificity of 87% neither of which was significantly different from cytology alone. There were three false positive cases on cytology and 13 false positive cases on NMP22 assay. There were three false negative cytology and five false negative NMP22 cases but only one was false negative for both, resulting in a high sensitivity (96%) but low specificity (30%) if either positive NMP22 or malignant or suspicious cytology was taken as a positive result. CONCLUSION: Combining NMP22 with malignant or suspicious cytological result improved sensitivity for the detection of bladder cancer but with a major decrease in specificity, suggesting a potential role in screening rather than diagnosis.

Raf kinase inhibitor protein: mechanism of loss of expression and association with genomic instability.

AIMS: Raf kinase inhibitory protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is an endogenous inhibitor of the Raf- MAPK kinase (MEK)-MAP kinase pathway. It has emerged as a significant metastasis suppressor in a variety of human cancers including colorectal cancer (CRC) and was recently shown to regulate the spindle checkpoint in cultured cells. This study aims at correlating RKIP expression with chromosomal instability in colorectal cancer samples and identifies possible mechanisms of RKIP loss. METHODS: Chromosomal instability was assessed using metaphase-based comparative genomic hybridisation (CGH) and loss of heterozygosity (LOH) in 65 cases with microsatellite stable CRC and correlated with RKIP expression. Methyl-specific PCR was used on DNA extracted from 82 cases with CRC to determine CpG methylation status at the RKIP promoter and the results correlated with RKIP protein expression. RESULTS: We demonstrate for the first time that in microsatellite stable (MSS) CRC, the number of chromosomal losses is inversely proportional to RKIP expression levels. We also show that methylation of the RKIP promoter is a major mechanism by which RKIP expression is silenced in CRC. CONCLUSIONS: RKIP loss by hypermethylation of its promoter could have a significant influence on colorectal cancer aneuploidy, which might explain its association with metastatic progression.

Metastatic recurrence of early-stage colorectal cancer is linked to loss of heterozygosity on chromosomes 4 and 14q.

OBJECTIVE: To investigate the prognostic value for loss of heterozygosity (LOH) of chromosomes 4 and 14q in early-stage colorectal cancer (CRC). METHODS: A total of 70, largely microsatellite stable, tumours and their corresponding normal mucosa were subjected to microdissection and analysed for LOH at chromosomes 4 and 14q by using 13 highly polymorphic microsatellite markers. LOH was correlated with the survival of the patients, using univariate, multivariate and Kaplan-Meier's survival curves. RESULT: LOH at D4S2935, D4S1579 and D4S1595 on chromosome 4 was significantly associated with metastatic recurrence of early-stage CRC. For chromosome arm 14q, two minimal regions of deletion were associated with metastatic recurrence and mapped to neighbouring markers D14S275/D14S49 at 14q12-13 and D14S65/D14S250 at 14q32. High-level loss (loss of five to eight of the informative microsatellite markers) on both chromosomes 4 and 14q, to be an independent prognostic indicator in early-stage CRC was shown by multivariate analysis. CONCLUSION: Determining the LOH of chromosomes 4 and 14q and their extent in primary tumours of patients with early-stage CRC may constitute a molecular signature of metastatic recurrence. This may be achieved if new finding sheds light on the treatment of this subgroup of patients that have been largely ignored.

BRCA1 gene expression in breast cancer in Kuwait: correlation with prognostic parameters.

OBJECTIVES: This study assessed the BRCA1 gene expression in breast cancer in Kuwait, and compared it with other known prognostic factors for the disease. MATERIALS AND METHODS: Forty-eight random samples of archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression. Immunohistochemical method utilizing antibodies against different epitopes on the BRCA1 protein was used to study BRCA1 protein expression. In addition, for 29 patients, reverse transcription-polymerase chain reaction was used to detect BRCA1 mRNA expression. BRCA1 expression was correlated with age, histological type and grade of breast cancer, estrogen and progesterone receptor status, and C-erbB-2 expression. RESULTS: No demonstrable BRCA1 mRNA and protein expression was found in 79 and 83% of the breast cancer tissues, respectively. A positive relationship was demonstrated between lack of BRCA1 (mRNA and protein) expression and high histological grade, negative estrogen and progesterone receptor status, and overexpression of C-erbB-2 in the breast cancer tissues. CONCLUSIONS: The study demonstrated lack of BRCA1 gene expression (mRNA and protein) in the majority of breast cancers in Kuwait and confirmed the inverse relationship between BRCA1 expression and parameters that determine poor prognosis in breast cancer.

Alpha-lipoic acid mitigates insulin resistance in Goto-Kakizaki rats.

Impaired glucose uptake and metabolism by peripheral tissues is a common feature in both type I and type II diabetes mellitus. This phenomenon was examined in the context of oxidative stress and the early events within the insulin signalling pathway using soleus muscles derived from non-obese, insulin-resistant type II diabetic Goto-Kakizaki (GK) rats, a well-known genetic rat model for human type II diabetes. Insulin-stimulated glucose transport was impaired in soleus muscle from GK rats. Oxidative and non-oxidative glucose disposal pathways represented by glucose oxidation and glycogen synthesis in soleus muscles of GK rats appear to be resistant to the action of insulin when compared to their corresponding control values. These diabetes-related abnormalities in glucose disposal were associated with a marked diminution in the insulin-mediated enhancement of protein kinase B (Akt/PKB) and insulin receptor substrate-1 (IRS-1)-associated phosphatidylinostol 3-kinase (PI 3-kinase) activities; these two kinases are key elements in the insulin signalling pathway. Moreover, heightened state of oxidative stress, as indicated by protein bound carbonyl content, was evident in soleus muscle of GK diabetic rats. Chronic administration of the hydrophobic/hydrophilic antioxidant alpha -lipoic-acid (ALA, 100 mg/kg, i.p.) partly ameliorated the diabetes-related deficit in glucose metabolism, protein oxidation as well as the activation by insulin of the various steps of the insulin signalling pathway, including the enzymes Akt/PKB and PI-3 kinase. Overall, the current investigation illuminates the concept that oxidative stress may indeed be involved in the pathogenesis of certain types of insulin resistance. It also harmonizes with the notion of including potent antioxidants such as ALA in the armamentarium of antidiabetic therapy.

Expressive genomic hybridisation: gene expression profiling at the cytogenetic level.

AIMS: To describe a cytogenetic technique suitable for the rapid assessment of global gene expression that is based on comparative genomic hybridisation (CGH), and to use it to understand the relation between genetic amplifications and gene expression. METHODS: Whereas traditional CGH uses DNA as test and reference in hybridisations, expressive genomic hybridisation (EGH) uses globally amplified mRNA as test and normal DNA as reference. EGH is a rapid and powerful tool for localising and studying global gene expression profiles and correlating them with loci of genetic amplifications using traditional CGH. RESULTS: EGH was used to correlate genetic amplifications detected by CGH with the expression profile of two independent cell lines-Colo320 and T47D. Although many amplifications resulted in overexpression, other amplifications were partially or completely silenced at the cytogenetic level. CONCLUSION: This technique will assist in the analysis of overexpressed genes within amplicons and could resolve a controversial issue in cancer cytogenetics; namely, the relation between genetic amplifications and overexpression.

Differences in in vitro invasive capacity induced by differences in Ki-Ras protein mutations.

The p21 proteins encoded by N-, Ki-, and H-ras are small guanine nucleotide-binding proteins that act as switches in several signal transduction pathways. Recently, evidence has been accumulating to suggest that valine-12 mutation in the Ki-Ras protein is associated with lung and colorectal tumours that are more aggressive than those carrying aspartate-12 mutation. The purpose of this study was to determine whether cells transfected with different Ki-ras codon-12 mutants have different biological behaviours in vitro that could reflect the differences in behaviour in vivo. For that reason, Rat-1 fibroblasts transfected with the valine-12 or aspartate-12 mutant or the wild-type Ki-ras gene were assessed in terms of in vitro invasion, transformation, and VEGF production. Both mutants demonstrated equal abilities to transform Rat-1 cells and induce VEGF production, while cells transfected with wild-type Ki-Ras failed to do so. Most significantly, the valine-12 mutants demonstrated a greater ability to invade Matrigel than cells expressing the aspartate-12 mutant or wild-type Ki-Ras proteins. This study complements previous experimental data that specific Ras mutations differ in their effects in vivo and shows, for the first time, a significant difference in Matrigel invasion in vitro. The precise mechanisms behind these biological differences in vivo and in vitro should now be investigated.

Carbonyl reductase as a significant predictor of survival and lymph node metastasis in epithelial ovarian cancer.

We have recently reported a novel function for carbonyl reductase (CR), namely, its ability to modulate the metastatic potential of malignant mouse cells. Because there are currently no data addressing a similar function for CR in human cancers, the aim of this study was to assess a correlation between survival and metastasis, and CR level in epithelial ovarian cancer. Using anti-CR antibody, immunohistochemical staining was performed on 73 epithelial ovarian cancers, 13 borderline malignant tumours, and 25 benign ovarian tumours for a total of 111 specimens. The combined rate for strongly and weakly positive reactions for CR was 32.0% for benign tumours, 38.5% for borderline malignant tumours, and 61.6% for ovarian cancers. The CR-positive rate was 35.7% (weakly positive alone) for ovarian cancers with retroperitoneal lymph node (RLN) metastasis and 67.8% for those without RLN metastasis (P< 0.05). The 5-year survival rate was 62.7% for the patients with CR-negative cancer and 86.1% for those with CR-positive cancer (P< 0.05). The present results indicate that decreased CR expression in epithelial ovarian cancer is associated with RLN metastasis and poor survival.

Carbonyl reductase: a novel metastasis-modulating function.

To explore reasons for differences in the malignancy of tumors, we have compared two cell lines derived from a mouse lung adenocarcinoma cell line that differ 10-fold in their capacity to form lung metastases from s.c. primary tumors or after i.v. injection. One mRNA encoding carbonyl reductase was identified at a relatively high abundance in the subline with low metastatic capacity but was not detectable in the highly metastatic subline. Transfection of the former subline with a plasmid construct expressing antisense carbonyl reductase rendered the cells highly metastatic. Conversely, the capacity of the highly metastatic cells to metastasize was markedly reduced after transfection with a construct expressing carbonyl reductase. We also found that human prostate cancers show loss of carbonyl reductase expression compared with normal prostate epithelia. These data suggest that carbonyl reductase has an important function in modifying the metastatic behavior of malignant tumors.

Treatment of hyperthyroidism in Down syndrome: case report and review of the literature.

Thyroid disorders are common in individuals with Down syndrome (DS). Hyperthyroidism occurs much less frequently than hypothyroidism in this population, but is likely to be underestimated. We report a case of an institutionalized adult male with DS and hyperthyroidism. He was treated with radioactive iodine and, when reviewed 11 weeks later, was found to be markedly hypothyroid. We also review the literature on the three treatment options for hyperthyroidism in DS: surgery, medical treatment, and radiotherapy. We concluded that the place of radioiodine in the treatment of hyperthyroid patients with DS is yet to be defined.

Structural differences between valine-12 and aspartate-12 Ras proteins may modify carcinoma aggression.

Recent evidence associates the codon 12 valine-for-glycine (G12V) mutant Ki-Ras protein with higher stage and increased lethality of colorectal carcinomas, while the codon 12 aspartate-for-glycine (G12D) Ras mutation shows no such association. Several observations may be relevant to this phenomenon. First, GTPase activity of G12V Ras is one-quarter that of G12D Ras and one-tenth that of wild-type (WT) Ras. Second, binding of the GTP analogue GppNp to G12D Ras is 8-fold weaker than its binding to G12V or WT Ras and crystal structures indicate that electrostatic repulsion between the carboxylate group of the G12D Asp-12 side-chain and the gamma phosphate of the bound nucleotide may make GTP binding to G12D Ras weaker even than that of GppNp. It is proposed that this lowering of affinity for GTP allows G12D Ras an escape from the oncogenic GTP-bound state, whereas GTP tightly bound to G12V mutant Ras generates a more persistent, potentially oncogenic, signal. Structural comparisons also suggest that differences between the Switch I (effector) region of G12D and G12V Ras could modify interactions with downstream signalling molecules such as Raf-1, neurofibromin, and phosphatidylinositol 3-hydroxy-kinase. Other differences between the G12D and G12V mutant Ras proteins include a lower affinity of the GTPase activating protein GAP for G12V than for G12D or WT Ras; but, as both G12D and G12V Ras are refractory to GTPase activation by GAP binding, this may be less significant. These studies complement experimental data showing that such Ras mutations differ in their effects in vitro and in vivo and, with recent data indicating heterogeneity of ras mutation in colorectal carcinomas and other tumours, make it plausible that codon 12 Ras mutations differ in carcinogenic potential and prognostic significance.

Comparative genomic hybridization analysis of primary colorectal carcinomas and their synchronous metastases.

We have analyzed 26 tumors from 12 patients with metastatic colorectal adenocarcinoma by comparative genomic hybridization (CGH). Primary tumors and their lymph node metastases from five Dukes' C patients as well as primary tumors and their liver metastases from seven Dukes' D patients were used to assess the extent of genetic differences between primary and secondary colorectal carcinomas from the same patients, to calculate the degree of clonal divergence and genetic heterogeneity in metastatic colorectal cancer, and to determine the differences in genetic imbalances between Dukes' C and D stage tumors. We show that the same genetic aberrations were frequently found in the primary tumors and their metastases. However, metastases often contained genetic aberrations not found in the corresponding primary tumors. The comparison of Dukes' stages C and D revealed genetic aberrations common to both. However, reduced copy number of chromosome arm 17p (5/5 vs. 0/7; P = 0.001) was significantly associated with Dukes' stage C and lymph node metastases, while increased copy number of chromosome arms 6p (6/7 vs. 0/5; P = 0.007) and 17q (5/7 vs. 0/5; P = 0.027) was associated more with Dukes' stage D and liver metastases. Our results established a repertoire of chromosomal alterations associated with metastatic colorectal cancer and suggest that Dukes' C (lymph node metastasis) tumors are not always simply an earlier stage of Dukes' D (liver metastasis) tumors and, thus, in some instances at least, they are distinct forms of the disease.

Hemizygosity of the MAX gene locus in HL60 cells.

The oncogenic activity of c-MYC is well known, and genetic aberrations in this locus are associated with a variety of human neoplasms. Because the encoded MYC protein has transcriptional activity only when dimerized with MAX, it is possible that mutations of MAX also could have phenotypic consequences. We have now found, by fluorescence in situ hybridization and quantified Southern blot analyses, that the MAX gene has been reduced to hemizygosity in HL60 cells. Although the sequence of the coding region of the remaining allele of the MAX gene is not mutated, this reduction in gene dosage may be the cause of a lower abundance of MAX protein in these cells that could result in an imbalance in the complex transcription factor network in which MAX has a pivotal role.

Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality.

The point mutations occurring in codons 12 and 13 of Ki-ras in 78 patients with colorectal carcinoma (31 Dukes' A and B, 21 Dukes' C, and 26 Dukes' D) have been determined by allele-specific oligonucleotide hybridization and sequencing. Duplicate samples of invasive primary carcinoma, adjacent normal tissue, and available lymph node and liver metastases from the same patients were microdissected from paraffin sections. There were no differences in the mutation rate between primary carcinomas and secondary deposits: 26 of 78 (33 per cent) primary carcinomas, 10 of 32 (31 per cent) lymph node metastases, and 10 of 26 (38 per cent) liver metastases. Multiple sampling revealed frequent heterogeneity within carcinomas: 9 of 26 primaries with Ki-ras mutations also contained areas of carcinoma with only the wild-type gene, implying that Ki-ras mutation, even when present in a colonic carcinoma, may not have been necessary for establishing the malignant phenotype. Also, 2 of 26 (8 per cent) Dukes' D patients had a mutation in their primary carcinoma but none in liver metastases and 6 of 47 (13 per cent) Dukes' C and D patients had mutations in liver or lymph node metastases but none in the primary carcinoma. Such heterogeneity may modify the effectiveness of novel therapies targeting mutant Ki-ras function, such as farnesyltransferase inhibition. Mutation of codon 12 from GGT (glycine) to GTT (valine) was more prevalent in primary and metastatic deposits of Dukes' C/D carcinomas (P = 0.01) than in primary carcinomas from Dukes' A/B patients. Mutations of codon 12 to GAT, AGT, GCT and codon 13 GGC to GAC were also found, but no correlation with carcinoma aggressiveness was apparent. Follow-up of 71/78 patients (up to 12 years) revealed decreased overall survival (P = 0.001) in patients with the GGT to GTT transversion in codon 12, even when the analysis was restricted to Dukes' D cases, supporting the suggestion that this mutation may confer a more aggressive phenotype in colorectal carcinoma.