AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling.

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Aryl hydrocarbon receptor interacting protein mutations seem not to associate with familial non-medullary thyroid cancer.

BACKGROUND: Over 95% of all thyroid malignancies are non-medullary thyroid carcinomas (NMTC). Familial NMTC are more aggressive and mortality is higher as compared with sporadic carcinomas. Known genetic factors do not explain all familial NMTC. Recently, thyroid disorders have been observed in families with germline mutations in aryl hydrocarbon receptor interacting protein (AIP) but, due to frequent occurrence of these conditions in the population, the significance of this co-occurrence is not clear. AIM, SUBJECTS AND METHODS: To examine whether AIP is involved in familial NMTC, we performed AIP mutation screening in 93 familial NMTC cases. In addition, the AIP status was studied in one follicular thyroid adenoma patient with a known AIP mutation from an additional cohort. RESULTS: No potentially pathogenic changes were identified, but two likely rare polymorphisms were detected. AIP mutation-positive patient's follicular thyroid adenoma showed no loss of heterozygosity or lack of immunohistochemical AIP staining. CONCLUSION: Our study indicates that germline AIP mutations are rare or do not exist in familial NMTC.

Mechanically-biologically treated municipal solid waste as a support medium for microbial methane oxidation to mitigate landfill greenhouse emissions.

The residual fraction of mechanically-biologically treated municipal solid waste (MBT residual) was studied in the laboratory to evaluate its suitability and environmental compatibility as a support medium in methane (CH(4)) oxidative biocovers for the mitigation of greenhouse gas emissions from landfills. Two MBT residuals with 5 and 12 months total (aerobic) biological stabilisation times were used in the study. MBT residual appeared to be a favourable medium for CH(4) oxidation as indicated by its area-based CH(4) oxidation rates (12.2-82.3 g CH(4) m(-2) d(-1) at 2-25 degrees C; determined in CH(4)-sparged columns). The CH(4) oxidation potential (determined in batch assays) of the MBT residuals increased during the 124 d column experiment, from <1.6 to a maximum of 104 microg CH(4) g(dw)(-1) h(-1) (dw=dry weight) at 5 degrees C and 578 microg CH(4) g(dw)(-1) h(-1) at 23 degrees C. Nitrous oxide (N(2)O) production in MBT residual (<15 microg N(2)O kg(dw)(-1) d(-1) in the CH(4) oxidative columns) was at the lower end of the range of N(2)O emissions reported for landfills and non-landfill soils, and insignificant as a greenhouse gas source. Also, anaerobic gas production (25.6 l kg(dw)(-1) during 217 d) in batch assays was low, indicating biological stability of the MBT residual. The electrical conductivities (140-250 mS m(-1)), as well as the concentrations of zinc (3.0 mg l(-1)), copper (0.5 mg l(-1)), arsenic (0.3 mg l(-1)), nickel (0.1 mg l(-1)) and lead (0.1 mg l(-1)) in MBT residual eluates from a leaching test (EN-12457-4) with a liquid/solid (L/S) ratio of 10:1, suggest a potential for leachate pollutant emissions which should be considered in plans to utilise MBT residual. In conclusion, the laboratory experiments suggest that MBT residual can be utilised as a support medium for CH(4) oxidation, even at low temperatures, to mitigate greenhouse gas emissions from landfills.

No evidence of somatic aryl hydrocarbon receptor interacting protein mutations in sporadic endocrine neoplasia.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently observed in patients with pituitary adenoma predisposition (PAP). Though AIP mutation-positive individuals with prolactin-, mixed growth hormone/prolactin-, and ACTH-producing pituitary adenomas as well as non-secreting pituitary adenomas have been reported, most mutation-positive patients have had growth hormone-producing adenomas diagnosed at relatively young age. Pituitary adenomas are also component tumors of some familial endocrine neoplasia syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Carney complex (CNC). Genes underlying MEN1 and CNC are rarely mutated in sporadic pituitary adenomas, but more often in other lesions contributing to these two syndromes. Thus far, the occurrence of somatic AIP mutations has not been studied in endocrine tumors other than pituitary adenomas. Here, we have analyzed 32 pituitary adenomas and 79 other tumors of the endocrine system for somatic AIP mutations by direct sequencing. No somatic mutations were identified. However, two out of nine patients with prolactin-producing adenoma were shown to harbor a Finnish founder mutation (Q14X) with a complete loss of the wild-type allele in the tumors. These results are in agreement with previous studies in that prolactin-producing adenomas are component tumors in PAP. The data also support the previous finding that somatic AIP mutations are not common in pituitary adenomas and suggest that such mutations are rare in other endocrine tumors as well.

Mutation analysis of aryl hydrocarbon receptor interacting protein (AIP) gene in colorectal, breast, and prostate cancers.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers.

No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, mostly MLH1 and MSH2. Somatic inactivation of the wild-type allele of the respective MMR gene is required for tumor development. Unexpectedly, a recent study utilizing DNA from paraffin-embedded tissue material detected frequent loss of the mutant MMR gene allele in HNPCC tumors. Dual role for loss of heterozygosity (LOH) was proposed. If somatic loss of the wild-type MMR gene allele had occurred through point mutation or promoter hypermethylation, frequent somatic deletions at the region of the MMR gene locus, perhaps targeting other relevant cancer genes, could quite commonly lead to loss of the mutant allele. To test this hypothesis, we studied a population-based series of 25 fresh-frozen HNPCC tumors with a germline mutation in MLH1 or MSH2 for LOH. Fourteen of the 25 tumors (56%) showed LOH at the respective locus, and all 14 losses targeted the wild-type allele (P=0.00006). These results strongly support the traditional two-hit model of HNPCC gene inactivation.

Analysis of microsatellite variation in Pinus radiata reveals effects of genetic drift but no recent bottlenecks.

Most conifer species occur in large continuous populations, but radiata pine, Pinus radiata, occurs only in five disjunctive natural populations in California and Mexico. The Mexican island populations were presumably colonized from the mainland millions of years ago. According to Axelrod (1981), the mainland populations are relicts of an earlier much wider distribution, reduced some 8,000 years ago, whereas according to Millar (1997, 2000), the patchy metapopulation-like structure is typical of the long-term population demography of the species. We used 19 highly polymorphic microsatellite loci to describe population structure and to search for signs of the dynamics of population demography over space and time. Frequencies of null alleles at microsatellite loci were estimated using an approach based on the probability of identity by descent. Microsatellite genetic diversities were high in all populations [expected heterozygosity (H(e)) = 0.68-0.77], but the island populations had significantly lower estimates. Variation between loci in genetic differentiation (F(ST)) was high, but no locus deviated statistically significantly from the rest at an experiment wide level of 0.05. Thus, all loci were included in subsequent analysis. The average differentiation was measured as F(ST) = 0.14 (SD 0.012), comparable with earlier allozyme results. The island populations were more diverged from the other populations and from an inferred common ancestral gene pool than the mainland ones. All populations showed a deficiency of expected heterozygosity given the number of alleles, the mainland populations more so than the island ones. The results thus do not support a recent important contraction in the mainland range of radiata pine.

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in Peutz-Jeghers syndrome.

Mutations in LKB1 lead to Peutz-Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

Comparing EST-based genetic maps between Pinus sylvestris and Pinus taeda.

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F(1) progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.

Screening for microsatellite instability target genes in colorectal cancers.

BACKGROUND: Defects in the DNA repair system lead to genetic instability because replication errors are not corrected. This type of genetic instability is a key event in the malignant progression of HNPCC and a subset of sporadic colon cancers and mutation rates are particularly high at short repetitive sequences. Somatic deletions of coding mononucleotide repeats have been detected, for example, in the TGFbetaRII and BAX genes, and recently many novel target genes for microsatellite instability (MSI) have been proposed. Novel target genes are likely to be discovered in the future. More data should be created on background mutation rates in MSI tumours to evaluate mutation rates observed in the candidate target genes. METHODS: Mutation rates in 14 neutral intronic repeats were evaluated in MSI tumours. Bioinformatic searches combined with keywords related to cancer and tumour suppressor or CRC related gene homology were used to find new candidate MSI target genes. By comparison of mutation frequencies observed in intronic mononucleotide repeats versus exonic coding repeats of potential MSI target genes, the significance of the exonic mutations was estimated. RESULTS: As expected, the length of an intronic mononucleotide repeat correlated positively with the number of slippages for both G/C and A/T repeats (p=0.0020 and p=0.0012, respectively). BRCA1, CtBP1, and Rb1 associated CtIP and other candidates were found in a bioinformatic search combined with keywords related to cancer. Sequencing showed a significantly increased mutation rate in the exonic A9 repeat of CtIP (25/109=22.9%) as compared with similar intronic repeats (p< or =0.001). CONCLUSIONS: We propose a new candidate MSI target gene CtIP to be evaluated in further studies.

Rapid expansion of microsatellite sequences in pines.

Microsatellite persistence time and evolutionary change was studied among five species of pines, which included a pair of closely related species (Pinus sylvestris and Pinus resinosa) in the subgenus Pinus, their relative Pinus radiata, and another closely related species pair (Pinus strobus and Pinus lambertiana) in the subgenus Strobus. The effective population sizes of these species are known to have ranged from the very small bottlenecks of P. resinosa to vast populations of P. sylvestris. This background allowed us to place the microsatellite evolution in a well-defined phylogenetic setting. Of 30 loci originating from P. strobus and P. radiata, we were able to consistently amplify 4 in most of the these pine species. These priming sites had been conserved for over 100 Myr. The four microsatellites were sequenced in the five species. Flanking sequences were compared to establish that the loci were orthologous. All microsatellites had persisted in these species, despite very different population sizes. We found a recent microsatellite duplication: a closely related pair of loci in P. strobus, where the other four species had just one locus. On two independent occasions, the repeat area of this same microsatellite (locus RPS 105a/b) had grown from a very low repeat number to 15 or 17 in the last 10-25 Myr. Other parts of the same compound microsatellite had remained virtually unchanged. Locus PR 4.6 is known to be polymorphic in both P. radiata and P. sylvestris, but the polymorphism in the two species is due to different motifs. The very large pine genomes are highly repetitive, and microsatellite loci also occur as gene families.

Steroid-involved transcriptional regulation of human genes encoding prostatic acid phosphatase, prostate-specific antigen, and prostate-specific glandular kallikrein.

We have compared the steroid regulation of human genes encoding prostatic acid phosphatase (hPAP), prostate-specific antigen (hPSA), and prostate-specific glandular kallikrein (hK2) at the level of transcription. Reporter constructs of hPAP promoter covering the region -734/+467 were functional in both prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines in transient transfections. hPAP -231/+50 with eight identified transcription factor-binding sites showed the highest, and hPAP -734/+467 showed the lowest transcriptional activity in CV-1 cells. The hPAP promoter could not be induced with androgen, glucocorticoid, or progesterone, contrary to the hPSA (-620/+40) and hK2 (-493/+27) promoters in PC-3 cells cotransfected with the respective steroid receptor expression vector. Therefore, steroids cannot directly regulate hPAP gene expression via receptor binding to steroid response elements at -178 and +336, which have been shown to have androgen receptor-binding ability in vitro. Glucocorticoid was the most powerful activator of the hPSA construct at 10-nM steroid concentrations. On the contrary, glucocorticoid stimulation of the transcriptional activity of the hK2 construct was the weakest among the tested steroids. The results indicate that the steroid response elements in the proximal promoters of hPSA and hK2 genes are not androgen specific, offering the molecular basis for the expression of these genes outside the prostate in tissues containing steroid receptors.

Do molecular markers reflect patterns of differentiation in adaptive traits of conifers?

We have examined patterns of variation of several kinds of molecular markers (isozymes, RFLPs of ribosomal DNA and anonymous low-copy number DNA, RAPDs and microsatellites) and an adaptive trait [date of bud set in Scots pine (Pinus sylvestris L.)]. The study included Finnish Scots pine populations (from latitude 60°N to 70°N) which experience a steep climatic gradient. Common garden experiments show that these populations are adapted to the location of their origin and genetically differentiated in adaptive quantitative traits, e.g. the date of bud set in first-year seedlings. In the northernmost population, bud set took place about 21 days earlier than in the southernmost population. Of the total variation in bud set, 36.4% was found among the populations. All molecular markers showed high levels of within-population variation, while differentiation among populations was low. Among all the studied markers, microsatellites were the most variable (He=0.77). Differences between populations were small, GST was less than 0.02. Our study suggests that molecular markers may be poor predictors of the population differentiation of quantitative traits in Scots pine, as exemplified here by bud-set date.