Genetic architecture of mouse skin inflammation and tumour susceptibility.

Germline polymorphisms in model organisms and humans influence susceptibility to complex trait diseases such as inflammation and cancer. Mice of the Mus spretus species are resistant to tumour development, and crosses between M. spretus and susceptible Mus musculus strains have been used to map locations of genetic variants that contribute to skin cancer susceptibility. We have integrated germline polymorphisms with gene expression in normal skin from a M. musculus x M. spretus backcross to generate a network view of the gene expression architecture of mouse skin. Here we demonstrate how this approach identifies expression motifs that contribute to tissue organization and biological functions related to inflammation, haematopoiesis, cell cycle control and tumour susceptibility. Motifs associated with inflammation, epidermal barrier function and proliferation are differentially regulated in backcross mice susceptible or resistant to tumour development. The intestinal stem cell marker Lgr5 is identified as a candidate master regulator of the hair follicle, and the vitamin D receptor (Vdr) is linked to coordinated control of epidermal barrier function, inflammation and tumour susceptibility.

miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells.

BACKGROUND: Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells. METHODS: We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting. RESULTS: Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100 beta-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins. CONCLUSION: microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.

Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer.

PURPOSE: This study was designed to elucidate the role of amplification at 8q24 in the pathophysiology of ovarian and breast cancer because increased copy number at this locus is one of the most frequent genomic abnormalities in these cancers. EXPERIMENTAL DESIGN: To accomplish this, we assessed the association of amplification at 8q24 with outcome in ovarian cancers using fluorescence in situ hybridization to tissue microarrays and measured responses of ovarian and breast cancer cell lines to specific small interfering RNAs against the oncogene MYC and a putative noncoding RNA, PVT1, both of which map to 8q24. RESULTS: Amplification of 8q24 was associated with significantly reduced survival duration. In addition, small interfering RNA-mediated reduction in either PVT1 or MYC expression inhibited proliferation in breast and ovarian cancer cell lines in which they were both amplified and overexpressed but not in lines in which they were not amplified/overexpressed. Inhibition of PVT1 expression also induced a strong apoptotic response in cell lines in which it was overexpressed but not in lines in which it was not amplified/overexpressed. Inhibition of MYC, on the other hand, did not induce an apoptotic response in cell lines in which MYC was amplified and overexpressed. CONCLUSIONS: These results suggest that MYC and PVT1 contribute independently to ovarian and breast pathogenesis when overexpressed because of genomic abnormalities. They also suggest that PVT1-mediated inhibition of apoptosis may explain why amplification of 8q24 is associated with reduced survival duration in patients treated with agents that act through apoptotic mechanisms.

Prognostic value of PAI1 in invasive breast cancer: evidence that tumor-specific factors are more important than genetic variation in regulating PAI1 expression.

Plasminogen activator inhibitor-1 (PAI1) can promote cancer progression, and its protein expression in tumors is an independent indicator of poor prognosis in many forms of cancer. Here, we show that high PAI1 mRNA levels also predict for shorter overall survival in two independent breast cancer data sets, highlighting the importance of its transcriptional regulation. The -675insG (4G/5G) single-nucleotide polymorphism in the PAI1 gene promoter has been shown to influence PAI1 transcription, with the 4G allele eliciting higher reporter gene expression in vitro and higher levels of circulating PAI1 in vivo. Nevertheless, its genotypic distribution in 2,539 British women with invasive breast cancer was virtually identical to that seen in 1,832 matched controls (P = 0.72), and annual mortality rates for 4G4G, 4G5G, and 5G5G cases were 2.6%, 2.8%, and 3.1% per year, respectively (P = 0.10). Thus, there was no association with breast cancer incidence or outcome, and in a separate set of breast cancers, the 4G/5G single-nucleotide polymorphism showed no association with PAI1 mRNA expression (P = 0.85). By contrast, connective tissue growth factor (CTGF), which can regulate PAI1 expression in culture, was associated with PAI1 expression in three independent cohorts (P << 0.0001). In addition, PAI1 gene copy number differences in the tumors were correlated with PAI1 mRNA expression (P = 0.0005) and seemed to affect expression independently of CTGF. Thus, local factors, such as CTGF and genomic amplification, seem to be more important than germ line genetic variation in influencing PAI1 expression and its untoward effects in breast cancer.

Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

CONTEXT: Matricellular proteins are a group of secreted, multifunctional extracellular matrix glycoproteins that includes thrombospondins (TSPs), tenascin-C, and secreted protein acidic and rich in cysteine (SPARC). They may be implicated in the dynamic developmental processes of the human fetal adrenal (HFA) in which the outer, definitive zone (DZ) cells are postulated to proliferate, migrate centripetally, differentiate, and populate the inner, steroidogenic fetal zone (FZ). OBJECTIVE: The objective of the study was to identify a matricellular molecule that likely plays a major role in HFA development. DESIGN: Studies involved RNA, cryosections, and cell cultures from 14- to 23-wk HFAs and human adult adrenal RNA. MAIN OUTCOME MEASURES: Measures included transcripts encoding matricellular proteins, using real-time quantitative RT-PCR; SPARC localization by immunostaining; and ACTH regulation of SPARC expression and secretion by quantitative RT-PCR and Western blot. RESULTS: SPARC HFA mRNA was 100-, 700-, and 300-fold higher than TSP-1, TSP-2, and tenascin-C mRNA, respectively. HFA SPARC mRNA was 3-fold higher than adult adrenals (P < 0.005), comparable with levels in adult brain (positive control), whereas mRNAs encoding TSP-1 and TSP-2 were lower in fetal than adult adrenals. SPARC immunoreactivity was detected exclusively in the FZ, not DZ. ACTH, a key regulator of HFA growth and function, increased SPARC mRNA (by 1.7-fold at 1 nm, 48 h, P < 0.05) in isolated FZ cells but not DZ cells. ACTH up-regulation of SPARC protein was also detected in FZ cell lysates and culture medium. CONCLUSIONS: Results suggest a possible role for SPARC in development of functional and/or structural zonation of the HFA.

Adrenocorticotropin preferentially up-regulates angiopoietin 2 in the human fetal adrenal gland: implications for coordinated adrenal organ growth and angiogenesis.

CONTEXT: ACTH is the key tropic hormone for the human fetal adrenal (HFA). Because vascular development must be coordinated with organ growth, ACTH may regulate local angiogenic factors, thereby influencing HFA angiogenesis. We previously demonstrated that ACTH up-regulates vascular endothelial growth factor in HFA cortical cells. A newer angiogenic factor family, the angiopoietins (Angs), also plays critical roles. Ang1 stabilizes, whereas Ang2 destabilizes vessels, increasing responsiveness to angiogenic stimuli. OBJECTIVE: The objective of this study was to investigate expression and ACTH regulation of Angs and their receptor Tie2 in the HFA. DESIGN, SETTING, AND PATIENTS: Studies were conducted involving RNA, frozen sections, and primary cell cultures from HFAs (14-24 wk) and human adult adrenal RNA. MAIN OUTCOME MEASURES: Angs and Tie2 mRNA levels were determined by real-time quantitative RT-PCR, Ang2 and Tie2 were localized by immunostaining, and ACTH regulation of Angs was investigated by real-time quantitative RT-PCR, Western blot, and ELISA. RESULTS: Mean HFA Ang2 to Ang1 mRNA ratio was 6.3-fold higher than adult adrenals (P < 0.001). Ang2 was localized predominantly in the HFA periphery, whereas Tie2 demonstrated endothelial localization. In isolated HFA cortical cells, ACTH up-regulated Ang mRNA levels in a time- and dose-dependent manner, with the balance favoring Ang2. Ang2 protein levels were elevated in ACTH-stimulated HFA cortical cells and conditioned medium. 8-Bromoadenosine cAMP and forskolin mimicked ACTH effects on the Angs. CONCLUSIONS: Higher HFA Ang2 to Ang1 ratios may reflect greater vascular remodeling than in the adult. Angs, particularly Ang2, in concert with vascular endothelial growth factor, may mediate ACTH tropic action, ensuring coordination of HFA growth, steroidogenesis, and angiogenesis.

Does familial non-medullary thyroid cancer adversely affect survival?

BACKGROUND: Familial non-medullary thyroid cancer (FNMTC) is associated with a higher rate of multifocality and a higher recurrence rate than sporadic thyroid cancer. However, the effect of FNMTC on life expectancy is unknown. MATERIAL AND METHODS: Using data from our FNMTC database, we calculated life expectancy and survival rates after diagnosis of FNMTC and compared the results with the rates for unaffected family members and for the standard US population. Overall life expectancy and survival rates were calculated using the Kaplan-Meier method. We compared patients from families with 2 affected members with patients from families with > or = 3 affected members. We also compared patients diagnosed in a known familial setting (index cases and subsequent cases) with patients diagnosed before the familial setting was recognized. RESULTS: There were 139 affected patients with 757 unaffected family members. The mean age at diagnosis was 40.8 +/- 13.9 years and the mean follow-up time was 9.4 +/- 11.7 years. Ten patients died of thyroid cancer during follow-up. The life expectancy of patients with FNMTC was similar to that of their unaffected family members. Survival was significantly shorter for patients with 3 or more affected family members, for patients diagnosed before the familial setting was recognized, and for patients with anaplastic cancer. CONCLUSIONS: Our results suggest that FNMTC may be more aggressive than sporadic thyroid cancer, particularly in families with 3 or more affected members. However, when recognized and treated appropriately, it does not significantly shorten the overall life expectancy of the affected patients.

Copy number aberrations in mouse breast tumors reveal loci and genes important in tumorigenic receptor tyrosine kinase signaling.

Receptor tyrosine kinase (RTK) signaling plays a key role in the development of breast cancer. Defining the genes and pathways in the RTK signaling network that are important regulators of tumorigenesis in vivo will unveil potential candidates for targeted therapeutics. To this end, we used microarray comparative genomic hybridization to identify and compare copy number aberrations in five mouse models of breast cancer induced by wild-type and mutated forms of oncogenic ErbB2 or the polyomavirus middle T antigen (PyMT). We observed distinct genomic alterations among the various models, including recurrent chromosome 11 amplifications and chromosome 4 deletions, syntenic with human 17q21-25 and 1p35-36, respectively. Expression of oncogenic Erbb2 (NeuNT) under control of the endogenous Erbb2 promoter results in frequent (85%) amplification at the Erbb2 locus with striking structural similarity to the human amplicon, resulting in overexpression of at least two of the genes, Erbb2 and Grb7. Chromosome 11 amplicons distal to Erbb2 arise in a model (DB) overexpressing a mutant variant of PyMT (Y315/322F) unable to activate phosphatidylinositol 3-kinase. These amplicons are not observed in DB hyperplasias or in tumors overexpressing wild-type PyMT and result in overexpression of Grb2 and Itgb4. Distal chromosome 4 deletions occur in a significantly higher proportion of Erbb2 than PyMT tumors and encompass 14-3-3sigma (Stratifin), which is expressed at low or undetectable levels in the majority of NeuNT tumors. Our studies highlight loci and genes important in the regulation of tumorigenic RTK signaling in mammary epithelial cells in vivo.

Troglitazone, the peroxisome proliferator-activated receptor-gamma agonist, induces antiproliferation and redifferentiation in human thyroid cancer cell lines.

Troglitazone is a potent agonist for the peroxisome proliferator-activated receptor-gamma (PPARgamma) that is a ligand-activated transcription factor regulating cell differentiation and growth. PPARgamma may play a role in thyroid carcinogenesis since PAX8-PPARgamma1 chromosomal translocations are commonly found in follicular thyroid cancers. We investigated the antiproliferative and redifferentiation effects of troglitazone in 6 human thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO82-1 (anaplastic) cell lines. PPARgamma was expressed variably in these cell lines. FTC-236 and FTC-238 had a rearranged chromosome at 3p25, possibly implicating the involvement of the PPARgamma encoding gene whereas the other cell lines did not. Troglitazone significantly inhibited cell growth by cell cycle arrest and apoptotic cell death. PPARgamma overexpression did not appear to be a prerequisite for a response to treatment with troglitazone. Troglitazone also downregulated surface expression of CD97, a novel dedifferentiation marker, in FTC-133 cells and upregulated sodium iodide symporter (NIS) mRNA in TPC-1 and FTC-133 cells. Our investigations document that human thyroid cancer cell lines commonly express PPARgamma, but chromosomal translocations involving PPARgamma are uncommon. Troglitazone, a PPARgamma agonist, induced antiproliferation and redifferentiation in thyroid cancer cell lines. PPARgamma agonists may therefore be effective therapeutic agents for the treatment of patients with thyroid cancer that fails to respond to traditional treatments.

The ATM gene is a target for epigenetic silencing in locally advanced breast cancer.

Several epidemiological studies on ataxia-telangiectasia families indicate that obligate ATM heterozygotes display an elevated risk for developing breast cancer. However, a molecular basis for a potential link between diminished ATM function and sporadic breast malignancy remains elusive. Here, we show that 78% (18 out of a panel of 23) of surgically removed breast tumors (stage II or greater) displayed aberrant methylation of the ATM proximal promoter region as judged by methylation-specific PCR. Aberrant methylation of the ATM promoter was independently confirmed in several tumors by bisulfite sequencing. Moreover, bisulfite sequencing indicated that this region of the genome is subject to dense methylation. Further, we found a highly significant correlation (P = 0.0006) between reduced ATM mRNA abundance, as measured by real-time RT-PCR, and aberrant methylation of the ATM gene promoter. These findings indicate that epigenetic silencing of ATM expression occurs in locally advanced breast tumors, and establish a link at the molecular level between reduced ATM function and sporadic breast malignancy.

Overexpression of matrix metalloproteinase-1 and -9 mRNA is associated with progression of oral dysplasia to cancer.

PURPOSE: Although an important risk factor for oral cancer is the presence of epithelial dysplasia, many lesions will not progress to malignancy. Matrix metalloproteinases (MMPs) are zinc-dependent proteinases capable of digesting various structural components of the extracellular matrix. Because MMPs are frequently overexpressed in oral squamous cell carcinoma (SCC), we hypothesized that they are also overexpressed in oral dysplasias; we also hypothesized that those dysplasias that progress to oral cancer express higher levels of MMPs than those lesions that do not progress. EXPERIMENTAL DESIGN: In this retrospective study, we examined changes in MMP-1, -2, and -9 mRNA expression using quantitative TaqMan reverse transcription-polymerase chain reaction in 34 routinely processed oral dysplasias and 15 SCCs obtained from 34 patients. After several years of close follow-up, 19 dysplasias progressed to oral SCC and 15 did not. RESULTS: Overall, MMP-1 mRNA was overexpressed (>2-fold) in 24 of 34 (71%) dysplasias and 13 of 15 (87%) oral SCCs. MMP-2 overexpression was seen in 11 of 34 (32%) dysplasias and 7 of 15 (47%) cancers; for MMP-9, overexpression was identified in 29 of 34 (85%) dysplasias and 15 of 15 (100%) cancers. MMP-1 and -9 levels were significantly higher in the SCCs compared with all oral dysplasias (P = 0.004 and P = 0.01, respectively). MMP-1 and -9 mRNA levels were significantly higher in the oral dysplasias that progressed to oral cancer compared with those that did not (P = 0.04 and P = 0.002, respectively). CONCLUSIONS: Levels of MMP-1 and -9 mRNA may be markers of malignant transformation of oral dysplasia to oral cancer.

Genome-scale expression profiling of Hutchinson-Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease with widespread phenotypic features resembling premature aging. HGPS was recently shown to be caused by dominant mutations in the LMNA gene, resulting in the in-frame deletion of 50 amino acids near the carboxyl terminus of the encoded lamin A protein. Children with this disease typically succumb to myocardial infarction or stroke caused by severe atherosclerosis at an average age of 13 years. To elucidate further the molecular pathogenesis of this disease, we compared the gene expression patterns of three HGPS fibroblast cell strains heterozygous for the LMNA mutation with three normal, age-matched cell strains. We defined a set of 361 genes (1.1% of the approximately 33,000 genes analysed) that showed at least a 2-fold, statistically significant change. The most prominent categories encode transcription factors and extracellular matrix proteins, many of which are known to function in the tissues severely affected in HGPS. The most affected gene, MEOX2/GAX, is a homeobox transcription factor implicated as a negative regulator of mesodermal tissue proliferation. Thus, at the gene expression level, HGPS shows the hallmarks of a developmental disorder affecting mesodermal and mesenchymal cell lineages. The identification of a large number of genes implicated in atherosclerosis is especially valuable, because it provides clues to pathological processes that can now be investigated in HGPS patients or animal models.

CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation.

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-kappaB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-beta. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and beta(3) integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.

Diagnostic and prognostic value of fas and telomeric-repeat binding factor-1 genes in adrenal tumors.

It is often difficult to distinguish histologically between an adrenal cortical cancer and a benign adenoma, or to predict the prognosis of patients with adrenal cortical cancers. In this investigation, we examined whether apoptosis-regulating genes, bcl-xL and fas, and a telomere-related gene, telomeric-repeat binding factor-1 (TRF-1), differ between adrenal cortical cancers and benign adrenal tumors. Tissues from 4 adrenal cortical cancers were compared with 7 normal adrenal tissues, 17 cortical adenomas, 4 cortical hyperplasias, and 20 pheochromocytomas for expressions of bcl-xL and fas by RT-PCR, and for expressions of TRF-1 by real-time quantitative RT-PCR. All benign adrenal tissues expressed both the antiapoptosis gene, bcl-xL, and proapoptosis gene, fas, but the adrenal cortical cancers expressed only bcl-xL and not fas. TRF-1 increased by more than 30-fold in the adrenal cortical cancers, compared with benign adrenal tissues, and inversely correlated with the prognosis of patients with the adrenal cortical cancers. This lack of expression of fas in adrenal cortical cancer may help to distinguish it from benign adrenal tumors. The level of TRF-1 expression may be helpful prognostically for patients with adrenal cortical cancers.

Expression of vascular endothelial growth factor-C in benign and malignant thyroid tumors.

In contrast to vascular endothelial growth factor (VEGF), which stimulates angiogenesis, VEGF-C is thought to stimulate lymphangiogenesis. The role of VEGF-C in thyroid cancer pathogenesis has not been clarified. One might expect a different pattern of VEGF-C expression in the various types of thyroid cancer because of their different means of metastases. In this investigation, we determined whether the differential expression of VEGF-C might explain the different propensity to lymph node metastasis in thyroid cancers. One hundred eleven normal and neoplastic thyroid tissues were analyzed by real-time quantitative PCR. Papillary thyroid cancers had a higher VEGF-C expression than other thyroid malignancies (P < 0.0005 ANOVA). Among the normal thyroid tissues from patients with malignant or benign thyroid diseases, there was no significant difference in VEGF-C expression. Paired comparison of VEGF-C expression between thyroid cancers and normal thyroid tissues from the same patients showed a significant increase of VEGF-C expression in papillary thyroid cancer (1.10 +/- 0.41 vs. 0.70 +/- 0.13; P = 0.001) and a significant decrease of VEGF-C expression in medullary thyroid cancer (0.11 +/- 0.13 vs. 0.78 +/- 0.29; P = 0.001). In contrast, there was no significant difference of VEGF-C expression between cancer and normal tissues in other types of thyroid cancer. In summary, VEGF-C expression is increased in papillary thyroid cancer, compared with paired normal thyroid tissues, but not in other thyroid cancers that are also prone to lymph node metastasis. The lymphangiogenic role of VEGF-C in thyroid cancers therefore appears to be complex and other factors are likely to be also involved.

Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression.

Although an important risk factor for oral cancer is the presence of epithelial dysplasia, most of these lesions will not progress to malignancy. Presently, for the individual patient with dysplasia, there are few reliable markers that may indicate the likelihood of progression to oral cancer. Cathepsin L is a lysosomal protease that degrades extracellular matrix material. Because cathepsin L is frequently overexpressed in oral squamous cell carcinoma (SCC) we hypothesized that it is also overexpressed in oral premalignancy and that premalignant lesions that progressed to oral cancer expressed higher levels of cathepsin L than those premalignant lesions that did not. In this retrospective pilot study we examined changes in cathepsin L expression at the mRNA level using quantitative TaqMan RT-PCR and at the protein level by immunohistochemistry in 33 routinely processed oral dysplastic lesions and 14 SCCs obtained from 33 patients. Sixteen of the dysplastic lesions progressed to oral SCC and 17 did not after several years of follow-up. Cathepsin L mRNA was overexpressed in 16/33 (48%) dysplastic lesions and in 9/14 (64%) oral SCC. Cathepsin L protein was also overexpressed in a large proportion of dysplasias and cancers. Overexpression was independent of dysplasia grade and identified in both those patients who progressed to oral SCC and in those who did not. Levels of cathepsin L mRNA and protein did not differ significantly in the progressing versus non-progressing dysplasias (P=0.27). However, cathepsin L mRNA and protein were significantly lower in the non-progressing dysplasias when compared to the oral cancers (P=0.03) but not in the progressing dysplasias suggesting a trend for dysplasias with overexpressed cathepsin L to be more likely to progress to oral cancer.

Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-A, angiopoietin-1, and angiopoietin-2.

During placental development, vessel formation occurs initially by vasculogenesis and subsequently by branching and nonbranching angiogenesis. We investigated vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang)-1 and -2 transcript profiles, and the protein products that they encode in placentas from normotensive pregnancies throughout pregnancy. In addition, we compared these genes in placentas from normotensive women and those with preeclampsia during the third trimester. Quantitative real-time PCR analysis demonstrated that VEGF-A and Ang1 mRNA increased in a linear pattern by 2.5 (not significant) and 2.8%/wk (P = 0.034), respectively, whereas Ang2 decreased logarithmically by 3.5%/wk (P = 0.0003). Ang2 mRNA was 400- and 100-fold higher than Ang1 and VEGF-A, respectively, in the first trimester and declined to 20-fold and 7-fold in the third. Ang2 protein (ELISA) decreased by 4.7%/wk (P = 0.0001), whereas Ang1 and VEGF-A were undetectable. In preeclampsia compared with normotensive pregnancy, only VEGF-A mRNA increased significantly, by 3-fold (P = 0.006). This increase may be related to low oxygen tension, as VEGF-A is up-regulated by hypoxia. In situ hybridization and immunohistochemical studies revealed that VEGF-A was localized in cyto- and syncytiotrophoblast and perivascular cells, whereas Ang1 and Ang2 were only in syncytiotrophoblast and perivascular cells in the immature intermediate villi during the first and second trimesters, and mature intermediate and terminal villi during the third trimester. These data suggest that these molecules may play important roles in placental biology and chorionic villus vascular development and remodeling in an autocrine/paracrine manner. The tight correlation between Ang2 mRNA and protein indicates that regulation of placental vascular development occurs at the transcriptional, and not translational, level.

Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses.

Increasingly, there is the need to analyze gene expression in tumor tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from formalin-fixed, paraffin-embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of formalin fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of formalin fixation for 24, 48, and 72 hours and for 1 week in normal (2), oral epithelial dysplasia (3), and oral squamous cell carcinoma (4) specimens yielding 9 fresh and 36 formalin-fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-1, p21, and vascular endothelial growth factor (VEGF) in 15 randomly selected and routinely processed oral carcinomas. We were able to extract RNA suitable for quantitative reverse transcription (RT) from all fresh (9/9) and formalin-fixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected oral squamous cell carcinoma. We found that prolonged formalin fixation (>48 h) had a detrimental effect on quantitative RT polymerase chain reaction results that was most marked for MMP-1 and VEGF but less evident for p21 and EGFR. Comparisons of quantitative RT polymerase chain reaction and immunohistochemistry showed that for all markers, except p21, there was good correlation between mRNA and protein levels. p21 mRNA was overexpressed in only one case, but protein levels were elevated in all but one tumor, consistent with the established translational regulation of p21. These results show that RNA can be reliably isolated from formalin-fixed, paraffin-embedded tissue sections and can produce reliable quantitative RT-PCR data. However, results for some markers are adversely affected by prolonged formalin fixation times.

Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream.

The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.

Real-time quantitative polymerase chain reaction: a potential tool for genetic analysis in neuropathology.

Since its introduction in the early- to mid-1980s, the polymerase chain reaction (PCR) has been modified and optimized for an increasing number of applications. Early on, the focus was on the amplification of a specific nucleic acid template into quantities amenable to identification and experimental manipulation. While this remains an important application, recent technology has allowed the use of PCR to accurately quantitate the amount of a specific nucleic acid template present in a complex sample. Rather than simply analyzing the final product amount following the course of sequential cycles of amplification, quantitative PCR allows one to measure the accumulation of PCR product during the course of the reaction ("real-time PCR"). Under the appropriate conditions the number of PCR cycles required for the accumulation of a specific amount of product (during the exponential phase of the reaction) is a reflection of the relative amount of nucleic acid template present in the sample under analysis. Real-time quantitative PCR allows one to analyze a relatively large number of samples in a short period of time, potentially allowing multiple markers to be applied on a sample within a time frame consistent with clinical settings. In this overview, we will highlight the uses of real-time quantitative PCR as a potential diagnostic tool in neuropathology, focusing on the analysis of CNS tumors.