Development of orthotopic tumour models using ultrasound-guided intrahepatic injection.

Mouse models of human diseases are an essential part of the translational pipeline. Orthotopic tumour mouse models are increasingly being used in cancer research due to their increased clinical relevance over subcutaneous xenograft models, particularly in relation to metastatic disease. In this study, we have developed orthotopic colorectal cancer liver metastases (CRCLM) and primary cholangiocarcinoma (CCA) models in BALB/c nude mice using minimally invasive ultrasound-guided intrahepatic injection. Due to its minimally invasive nature, the method reduced risk from surgical complications whilst being fast and easy to perform and resulted in measurable tumour volumes 1 to 3 weeks post-injection. Tumour volumes were monitored in vivo by weekly high-frequency ultrasound (HF-US) and/or twice weekly bioluminescence imaging (BLI) and confirmed with end-point histology. Take rates were high for human CRC cells (>73%) and for CCA cells (90%). We have demonstrated that this method reliably induces CRCLM and CCAs, in which tumour volume can be monitored throughout using HF-US and/or BLI. This provides a promising experimental tool for future testing of cancer therapeutics in an orthotopic model.

Both isoforms of ketohexokinase are dispensable for normal growth and development.

Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. The molecular determinants of this epidemiologic correlation are incompletely defined, but high-flux fructose catabolism initiated by ketohexokinase (Khk, fructokinase) is believed to be important. The Khk gene encodes two enzyme isoforms with distinctive substrate preferences, the independent physiological roles of which are unclear. To investigate this question, and for testing the importance of Khk in metabolic syndrome, isoform-selective genetic lesions would be valuable. Two deficiency alleles of the mouse Khk gene were designed. The first, Khk(3a), uses targeted "knock-in" of a premature termination codon to induce a selective deficiency of the minor Khk-A isoform, preserving the major Khk-C isoform. The second, the Khk(Δ) allele, ablates both isoforms. Mice carrying each of these Khk-deficiency alleles were generated and validated at the DNA, RNA, and protein levels. Comparison between normal and knockout animals confirmed the specificity of the genetic lesions and allowed accurate analysis of the cellular distribution of Khk within tissues such as gut and liver. Both Khk(3a/3a) and Khk(Δ/Δ) homozygous mice were healthy and fertile and displayed minimal biochemical abnormalities under basal dietary conditions. These studies are the first demonstration that neither Khk isoform is required for normal growth and development. The new mouse models will allow direct testing of various hypotheses concerning the role of this enzyme in metabolic syndrome in humans and the value of Khk as a pharmacological target.

Inferring relative proportions of DNA variants from sequencing electropherograms.

MOTIVATION: Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. RESULTS: We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. AVAILABILITY AND IMPLEMENTATION: QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. CONTACT: msjimc@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

Lack of interleukin-4 receptor alpha chain-dependent signalling promotes azoxymethane-induced colorectal aberrant crypt focus formation in Balb/c mice.

Interleukin (IL)-4 receptor (IL-4R) alpha chain-dependent signalling by IL-4 and IL-13 promotes tumour growth and metastasis in mouse models of colorectal cancer. However, the role of IL-4R alpha-dependent signalling during the early, pre-malignant stages of colorectal carcinogenesis has not been investigated. Therefore, we investigated the effect of deletion of the IL-4R alpha gene on azoxymethane-induced colorectal aberrant crypt focus (ACF) multiplicity and size in Balb/c mice. IL-4R alpha(-/-) mice developed significantly more ACFs [median 8, inter-quartile range (IQR) 4-11.5; n = 9] than wild-type (WT) animals (median 4, IQR 1-6; n = 9; p = 0.04, Mann-Whitney U-test). There were significantly higher levels of IL-4 in serum from azoxymethane- and sham-treated IL-4R alpha(-/-) mice than WT animals, but no difference in serum IL-13 levels. In the absence of functional IL-4Rs, IL-13 can also signal via the IL-13R alpha2 receptor, leading to induction of transforming growth factor (TGF) beta, which has pro-tumourigenic activity at early stages of intestinal tumourigenesis. We found that mucosal TGFbeta mRNA levels and intestinal epithelial cell TGFbeta immunoreactivity were significantly higher in IL-4R alpha(-/-) mice than in WT animals. In summary, IL-4R alpha-dependent signalling has a protective, anti-neoplastic role during the post-initiation phase of azoxymethane-induced colorectal carcinogenesis in Balb/c mice. Our data should prompt thorough investigation of the role of IL-4R alpha-dependent signalling during human colorectal carcinogenesis, particularly as antagonism of IL-4R signalling represents a therapeutic strategy for asthma and other allergic diseases.

Human menopausal and pregnant mare serum gonadotrophins in murine superovulation regimens for transgenic applications.

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F(1) hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.

Regulation of stromal cell cyclooxygenase-2 in the ApcMin/+ mouse model of intestinal tumorigenesis.

Cyclooxygenase-2 (Cox-2) is expressed predominantly by stromal cells in intestinal adenomas from the Apc(Min/+) mouse model of familial adenomatous polyposis. We investigated the mechanistic basis of stromal cell Cox-2 expression in Apc(Min/+) mouse adenomas, as well as Cox-2 expression and activity in histologically normal (HN) Apc(Min/+) mouse intestine, in order to gain further insights into regulation of Cox-2 as a potential chemoprevention target. Upregulation of Cox-2 in intestinal tumours is not an intrinsic feature of Apc(Min/+) macrophages as bone marrow-derived Apc(Min/+) macrophages did not exhibit an abnormality in Cox-2 expression or activity. Intestinal permeability to lactulose or mannitol was similar in Apc(Min/+) mice and wild-type littermates, implying that macrophage activation by luminal antigen is unlikely to explain stromal cell Cox-2 induction. Moreover, stromal cells exhibited differential expression of Cox-2 and inducible nitric oxide synthase, suggesting 'alternative' (M2) rather than 'classical' (M1) macrophage activation. Flow cytometric sorting of isolated stromal mononuclear cells (SMNCs), on the basis of M-lysozyme and specific macrophage marker expression, demonstrated that macrophages, neutrophils and non-myelomonocytic cells all contributed to lamina propria prostaglandin (PG) E(2) synthesis. However, the majority of PGE(2) synthesis by macrophages was via a Cox-2-dependent pathway compared with predominant Cox-1-derived PGE(2) production by non-myelomonocytic cells. SMNCs from HN Apc(Min/+) intestinal mucosa exhibited similar levels of Cox-2 mRNA and protein, but produced more Cox-2-derived PGE(2) than wild-type cells at 70 days of age. There was an age-dependent decline in PGE(2) synthesis by Apc(Min/+) SMNCs, despite tumour progression. These data suggest that other Cox-2-independent factors also control PGE(2) levels during Apc(Min/+) mouse intestinal tumorigenesis. Regulation of macrophage Cox-2 expression and other steps in PGE(2) synthesis (e.g. PGE synthase) are valid targets for novel chemoprevention strategies that could minimize or avoid systemic COX-2 inhibition.

Marker gene transfer into human haemopoietic cells using a herpesvirus saimiri-based vector.

Herpesvirus-based gene therapy vectors offer an attractive alternative to retroviral vectors because of their episomal nature and ability to accommodate large transgenes. Saimiriine herpesvirus 2 (HVS) is a prototypical gamma-2 herpesvirus that can latently infect numerous different cell types. A cosmid-generated HVS vector in which transforming genes have been deleted and the marker gene encoding enhanced green fluorescent protein (HVS-GFP) has been incorporated was evaluated for its potential to transduce CD34+ haemopoietic progenitors selected from cord blood. Expression of GFP could subsequently be readily detected in cells of the erythroid lineage in both CFU-GEMM assays and liquid differentiation cultures. These results confirm the potential of HVS as a candidate vector for gene therapy applications using primitive haemopoietic cells and suggest that it may be applicable to disorders affecting cells of the erythroid lineage.

Linkage/association study of a locus modulating total serum IgE on chromosome 14q13-24 in families with asthma.

BACKGROUND: A study was undertaken to validate a locus modulating total serum IgE levels on 14q13-24. METHODS: A linkage and association study was performed between total serum IgE and a panel of seven microsatellites which map to the 14q13-24 region in 69 families with asthma recruited from Leeds, UK. RESULTS: Non-parametric, multipoint, sib pair analysis showed no evidence of genetic linkage between the quantitative trait "log IgE" and any of the tested markers. However, a significant association was observed between locus D14S63 (14q23) and total serum IgE (p = 0.017). Allelic analysis showed an association between low total IgE and allele 157 of D14S63 (p = 0.01, OR = 0.63, 95% CI 0.44 to 0.90). Modelling of allele 157 genotypes as a continuous covariate indicated evidence of a significant inverse linear trend across the three genotypes where 157 homozygotes had the lowest mean log IgE (p = 0.045). Association of D14S63 with log IgE was confirmed in the analysis of a combined dataset of 53 families from Southampton, UK and the 69 families from Leeds (total 122 families). An association was observed at the locus level (p = 0.022) and the allelic level where allele 165 showed an association with high total IgE (p = 0.001, OR = 3.79, 95% CI 1.54 to 9.7) and allele 157 showed an association with low total IgE (p = 0.041, OR = 0.77, 95% CI 0.6 to 0.99). The transmission disequilibrium test was positive for allele 165 (p<0.05) and negative for allele 157 (p>0.05). CONCLUSIONS: Despite the lack of linkage, the findings of this study support the previous observation of a gene(s) at 14q23 that modulates total serum IgE.

Molecular biology of prostate cancer.

Development of any cancer reflects a progressive accumulation of alterations in various genes. Oncogenes, tumour suppressor genes, DNA repair genes and metastasis suppressor genes have been investigated in prostate cancer. Here, we review current understanding of the molecular biology of prostate cancer. Detailed understanding of the molecular basis of prostate cancer will provide insights into the aetiology and prognosis of the disease, and suggest avenues for therapeutic intervention in the future.

Overexpression of transcripts containing LINE-1 in the synovia of patients with rheumatoid arthritis.

OBJECTIVE: To identify novel diagnostic markers by comparing gene expression in rheumatoid (RA) and reactive arthritis (ReA) synovium. METHODS: Synovial biopsy specimens were obtained by needle arthroscopy from the knees of 10 patients with either RA or ReA. RNA was isolated from the biopsy specimens and cDNA synthesised for analysis using a customised cDNA macroarray. Confirmatory analysis was performed using in situ hybridisation on a second set of synovial samples. RESULTS: Two unique transcripts (ReXS1 and fibronectin) were consistently more abundant in ReA and three homologous transcripts were more abundant in RA. The latter all mapped within long interspersed nucleotide elements (LINE-1), that form one of the families of repetitive sequences in the human genome. CONCLUSIONS: The abundance of transcripts containing LINE-1 in the RA synovium may be an epiphenomenon or may have pathogenic significance. Further work is required to determine the identity of the full length transcript(s) before its use as a diagnostic marker in RA can be assessed.

Cytogenetic alterations in ovarian clear cell carcinoma detected by comparative genomic hybridisation.

Ovarian clear cell carcinoma (OCCC) accounts for a small but significant proportion of all ovarian cancers and is a distinct clinical and pathological entity. It tends to be associated with poorer response rates to chemotherapy and with a worse prognosis. Little is known about possible underlying genetic changes. DNA extracted from paraffin-embedded samples of 18 pure OCCC cases was analysed for genetic imbalances using comparative genomic hybridisation (CGH). All of the 18 cases showed genomic alterations. The mean number of alterations detected by CGH was 6 (range 1-15) indicating a moderate level of genetic instability. Chromosome deletions were more common than amplifications. The most prominent change involved chromosome 9 deletions in 10 cases (55%). This correlates with changes seen in other epithelial ovarian cancers. This deletion was confirmed using microsatellite markers to assess loss of heterozygosity (LOH) at four separate loci on chromosome 9. The most distinct region of loss detected was around the IFNA marker at 9p21 with 41% (11 out of 27 cases) LOH. Other frequent deletions involved 1p (five out of 18; 28%); 11q (four out of 18; 22%) and 16 (five out of 18; 28%). Amplification was most common at chromosome 3 (six out of 18; 33%); 13q (four out of 18; 22%) and 15 (three out of 18; 17%). No high-level amplifications were identified. These features may serve as useful prognostic indicators in the management of OCCC.

FcgammaRIIIA-158V and rheumatoid arthritis: a confirmation study.

OBJECTIVES: To develop a robust assay for genotyping the FcgammaRIIIA-158V/F polymorphism and to confirm the putative association between the FcgammaRIIIA-158V allele and rheumatoid arthritis (RA). METHODS: This allelic association study examined the FcgammaRIIIA-158V/F polymorphism for association with RA. A novel single-stranded conformational polymorphism assay was used to genotype 828 RA patients and 581 controls from the UK. RESULTS: The FcgammaRIIIA-158V allele was associated with both RA (P=0.02) and nodules (P=0.04). Individuals homozygous for this higher affinity allele had a significantly increased risk of RA (OR 1.53, 95% CI 1.08-2.18) and the development of nodules (OR 2.20, 95% CI 1.20-4.01). There was no evidence of an interaction with the shared epitope. CONCLUSIONS: We have developed a novel assay to genotype the FcgammaRIIIA-158F/V polymorphism and confirmed that homozygosity for the FcgammaRIIIA-158V allele is associated with UK Caucasian RA, particularly in those individuals with nodules, suggesting FcgammaRIIIA may play a role in determining disease severity or in the development of nodules per se.

Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma.

Survivin is unique for its expression in human malignancies but not in normal adult cells. It has been implicated in sensitisation to chemotherapy and as a prognostic marker in several common cancers. Immunohistochemistry for Survivin, P53 and BCL-2 expression as well as cell proliferative index (Ki-67) and apoptosis index (TUNEL) was conducted on 52 pancreatic and 12 ampullary adenocarcinomas. Survivin was detected in the cytoplasm of carcinoma cells in 46 (88%) of pancreatic tumours. P53 and BCL-2 were detected in 54% and 12% of pancreatic tumours, respectively. Proliferative index was 26.2+/-10.5% and apoptosis index was 1.38+/-0.69%. Prevalence of Survivin expression was significantly higher in P53-positive than in P53-negative cases (P=0.05) but was not associated with BCL-2 expression. Incrementally higher weighted scores of Survivin expression were associated with increased proliferative index (P=0.001). Furthermore, there was linear correlation between increased proliferative index and higher apoptosis index (P<0.001). Surprisingly, higher scores of Survivin expression were associated with increased apoptosis index (P=0.007). Survival characteristics were not influenced by Survivin, P53 or BCL-2 expression, apoptosis index or proliferative index. Ampullary carcinoma showed Survivin expression in 83% of cases. However, unlike pancreatic carcinoma, there was no correlation between Survivin and P53 expression or proliferative index. In conclusion, Survivin is expressed in the majority of pancreatic adenocarcinomas and correlates with both cellular proliferation and apoptosis. Molecular manipulation of Survivin expression may enhance chemotherapy and radiation therapy for pancreatic cancer.

A family study and the natural history of prenatally detected unilateral multicystic dysplastic kidney.

PURPOSE: We document the inheritance pattern of multicystic dysplastic kidney in 3 affected families and screen first-degree relatives of a cohort of children with prenatally detected multicystic dysplastic kidney for renal anomalies. The study also afforded an opportunity to document the natural history of prenatally detected multicystic dysplastic kidney. MATERIALS AND METHODS: We identified 3 families during clinical treatment of children with prenatally detected multicystic dysplastic kidneys. Other members of these families were evaluated with renal ultrasonography. For the family screening study index cases were identified from a fetal uropathy database. A total of 94 first-degree relatives (52 parents, 35 full siblings and 7 half siblings) of 29 children with prenatally detected multicystic dysplastic kidneys were studied with urinary tract ultrasonography, blood pressure measurement, urinalysis and plasma biochemistry. RESULTS: Two families had affected sibling pairs, 1 of which also had a half sibling with vesicoureteral reflux. The third family included 3 individuals with multicystic dysplastic kidney and 1 with renal agenesis thought to have resulted from involution of multicystic dysplastic kidney. This family is consistent with autosomal dominant inheritance with variable expressivity and reduced penetrance. In the screening study ultrasonography did not demonstrate significant renal anomalies in any of the 94 first-degree relatives of the multicystic dysplastic kidney index cases. Followup assessment of prenatally detected multicystic dysplastic kidneys in index cases demonstrated total involution in 52% at a median age of 6.5 years with no multicystic dysplastic kidney related morbidity. CONCLUSIONS: Multicystic dysplastic kidney can be familial but is most commonly a sporadic anomaly. Formal screening of relatives is not recommended. Followup data on a cohort of children with prenatally detected multicystic dysplastic kidney add further support to conservative management.

BCL10 in malignant lymphomas--an evaluation using fluorescence in situ hybridization.

BCL10 is a tumour suppressor gene originally cloned from a t(1;14)(p22;q32) breakpoint in a case of mucosa-associated lymphoid tissue (MALT) lymphoma. Translocations involving this gene, though uncommon, are sometimes encountered in MALT lymphomas. This gene is thought to play an important role in the development of malignant lymphomas. Fluorescence in situ hybridization (FISH) was therefore undertaken on 22 cases of malignant lymphoma of varying histology to establish the incidence of rearrangements involving the BCL10 gene. Initially, one case with a novel t(1;2)(p22;p12) translocation involving the BCL10 gene was identified, in a marginal zone lymphoma of the MALT type, and was reported elsewhere. Seven other cases were subsequently identified with abnormalities in the 1p region, including a translocation with a breakpoint in the 1p22 region in a case of lymphoblastic lymphoma. However, none of these involved the BCL10 gene. Mutation analysis of BCL10 was then performed on 57 cases of malignant lymphoma, including 17 MALT lymphomas, by single-strand conformational polymorphism (SSCP) analysis of tumour DNA. Tissue was obtained for mutation analysis for 12 of the 22 cases analysed by FISH. Selected cases with SSCP band shifts were further studied by direct sequencing. Polymorphisms were identified in eight cases, but no mutations of pathogenic significance were identified. Further RT-PCR and mutation analysis was performed on cDNAs from 12 cases (four MALT, seven diffuse large B-cell lymphoma, one Hodgkin's disease) in which DNA analysis had already been completed. This included the MALT lymphoma with the t(1;2)(p22;p12) rearrangement. Again, no mutations were identified in the coding sequence. This study confirms that rearrangements of the BCL10 gene are uncommon in lymphoma (1/22) and may be limited tothe MALT subtype of non-Hodgkin's lymphomas. It was also found that breakpoints or rearrangements in the 1p22 region do not necessarily involve the BCL10 gene. Moreover, the absence of mutations at both the DNA (0/60) and the mRNA (0/12) level indicates that this gene is not frequently inactivated by mutation, in those tumours in which it is not involved in translocations. Our findings suggest that the BCL10 gene is unlikely to have a frequent or key role in general lymphomagenesis.

Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone.

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3 approximately p23.2, 2q21 approximately q34, 3p12.3 approximately p14.1, 3p22 approximately p26, 3q, 12q13.2 approximately q22, 13q12 approximately q14, 17q21.3 approximately q23, 20q11.2 approximately q13.1 and 21q11.2 approximately q21 as well as the deletion of 6p21.1, 6p23 approximately p25, 7q11.1 approximately q31, 7q35 approximately q36, 11p15, 11q24, 13q33, 17p, 18q12 approximately q21.1, 19p, 19q13.3, 22q13.1 approximately q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the Apc(Min/+) mouse.

BACKGROUND & AIMS: The role of the inducible isoform of nitric oxide synthase (Nos2 or iNOS) in intestinal tumorigenesis is unclear. Conflicting data also exist regarding the ability of Nos2 to modulate expression and/or activity of cyclooxygenase 2 (Cox-2), which promotes intestinal tumorigenesis. Therefore, we determined the effect of a null Nos2 genotype on intestinal tumorigenesis and Cox-2 expression/activity in the Apc(Min/+) mouse model of familial adenomatous polyposis. METHODS: Apc(Min/+)Nos2(-/-) mice were generated by successive crosses between C57BL/6-Apc(Min/+) and C57BL/6-Nos2(tm1Lau) mice. Adenoma characteristics of age-matched Apc(Min/+)Nos2(+/+) and Apc(Min/+)Nos2(-/-) mice were compared. The level and cellular localization of Nos2 messenger RNA (mRNA) expression in Apc(Min/+)Nos2(+/+) mouse intestine was determined. Cox-2 expression and activity were measured in both intestinal tissue and bone marrow-derived macrophages in vitro. RESULTS: Apc(Min/+)Nos2(-/-) mice developed significantly more intestinal adenomas than Apc(Min/+)Nos2(+/+) littermates. Epithelial cell Nos2 mRNA expression was decreased in adenomas compared with histologically normal Apc(Min/+)Nos2(+/+) intestine. There was no significant difference in Cox-2 expression or activity in either intestine or bone marrow-derived macrophages from Apc(Min/+)Nos2(+/+) and Apc(Min/+)Nos2(-/-) animals. CONCLUSIONS: Nos2 plays an antineoplastic role in the Apc(Min/+) mouse model of familial adenomatous polyposis. Nos2 does not modulate Cox-2 expression or activity in the Apc(Min/+) mouse.

Expression of the Sonic Hedgehog receptor "PATCHED" in basal cell carcinomas and odontogenic keratocysts.

Basal cell carcinoma (BCC) is a common invasive skin lesion in Caucasians. Odontogenic keratocysts (OKs) are developmental, non-inflammatory oral cysts. They can be sporadic and/or multiple and are locally destructive. Basal cell naevus syndrome (BCNS) comprises both multiple BCCs and multiple OKs, in addition to several other systemic manifestations. The genetic defect underlying this autosomal dominant syndrome is a germ line mutation in the Sonic Hedgehog receptor PATCHED (PTCH) gene. For this study, a rabbit anti-peptide PTCH antiserum was produced. Immunohistochemistry procedures were performed using PTCH antibody and commercially produced GLI-1 antibody (downstream member in the hedgehog pathway) to stain 11 BCNS-OKs, eight sporadic OKs, two BCNS-BCCs, and six sporadic BCCs. Most of these lesions had been previously screened for PTCH mutation. Most BCCs (n=7) demonstrated moderate staining, with the heaviest staining in the outer palisading cell layer, except a BCNS-BCC which had mutation proximal to the sequence used for production of immunogenic peptide; this demonstrated only weak staining. Although moderate to heavy staining with PTCH antibody was demonstrated in the epithelium of both types of OK (n=19), a quite different pattern of staining of the basal cell layer was observed in the two patient groups. In BCNS, OK staining was heaviest in basal epithelial layers. In contrast, staining in non-BCNS odontogenic keratocysts was exclusively located in the superficial epithelial layers. Up-regulation of PTCH and GLI-1 protein was demonstrated in both BCCs and OKs. The pattern of PTCH expression matched the PTCH transcript pattern previously reported in BCCs and appeared sufficiently characteristic in OKs to allow differentiation between syndromic and non-syndromic cysts.