beta(2)microglobulin mRNA expression levels are prognostic for lymph node metastasis in colorectal cancer patients.

Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT-PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I-IV CRC patients with (n=20) and without (n=18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of beta(2)microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC)=0.85; 95% confidence interval (CI)=0.69-0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC=0.87; 95% CI=0.71-0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R(2) value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.

Structural properties of Friedreich's ataxia d(GAA) repeats.

The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAA.TTC)n, a sequence whose expansion is associated with Friedreich's ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272-274 nm over the temperature range of 5-50 degrees C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm) of d(GAA)15 was 40 degrees C. At 5 degrees C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35 degrees C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5 degrees C, unknown but partially base-paired at 37 degrees C, and an approximately random coil above 65 degrees C.

Trinucleotide repeats associated with human disease.

Triplet repeat expansion diseases (TREDs) are characterized by the coincidence of disease manifestation with amplification of d(CAG. CTG), d(CGG.CCG) or d(GAA.TTC) repeats contained within specific genes. Amplification of triplet repeats continues in offspring of affected individuals, which generally results in progressive severity of the disease and/or an earlier age of onset, phenomena clinically referred to as 'anticipation'. Recent biophysical and biochemical studies reveal that five of the six [d(CGG)n, d(CCG)n, (CAG)n, d(CTG)n and d(GAA)n] complementary sequences that are associated with human disease form stable hairpin structures. Although the triplet repeat sequences d(GAC)n and d(GTC)n also form hairpins, repeats of the double-stranded forms of these sequences are conspicuously absent from DNA sequence databases and are not anticipated to be associated with human disease. With the exception of d(GAG)n and d(GTG)n, the remaining triplet repeat sequences are unlikely to form hairpin structures at physiological salt and temperature. The details of hairpin structures containing trinucleotide repeats are summarized and discussed with respect to potential mechanisms of triplet repeat expansion and d(CGG.CCG) n methylation/demethylation.

Physiological processes revealed through an analysis of inborn errors.

To develop the significance of human inborn errors of membrane function in terms of their potential for the elucidation of physiological processes, an overview of the genetics of inherited metabolic disorders is first presented, concisely covering classical Mendelian inheritance and progressing to molecular applications currently being used in the isolation of a number of disease-causing genes. Second, approaches to the isolation of several genes implicated in inherited disorders of membrane function are discussed. Examples are drawn from studies of the Shaker/IA K+ channel mutants of Drosophila and human inborn errors in the low-density lipoprotein receptor and Kidd antigen/urea transporter. These examples illustrate the techniques of "reverse" genetics, restriction fragment-length polymorphism analysis, chromosomal walking, and cDNA library screening with oligonucleotides and antibodies. Finally, the need to develop a physiological functional analysis of the protein structures derived from isolated human disease-causing genes is presented as a necessary direction for future research.

The purine-rich trinucleotide repeat sequences d(CAG)15 and d(GAC)15 form hairpins.

The structures of single-stranded (ss) oligonucleotides containing (CAG)15 [ss(CAG)15] or (GAC)15 [ss(GAC)15] were examined. At 10 degrees C, the electrophoretic mobilites of the two DNAs were similar to ss(CTG)15, a DNA that forms a hairpin containing base paired and/or stacked thymines. At 37 degrees C in 50 mM NaCl, single-strand-specific P1 nuclease cleaved the G33-G36 phosphodiesters of ss(GAC)15, and the G32-A34, G35-C36 phosphodiesters of ss(CAG)15 (where the loop apex of both DNAs = A34). Electrophoretic mobility melting profiles indicated that the melting temperature (Tm) of ss(CAG)15 in low (approximately 1 mM Na+) ionic strength was 38 degrees C. In contrast, the Tm of ss(GAC)15 was 49 degrees C, a value similar to the Tm of ss(CTG)15. These results provide evidence that ss(GAC)15 and ss(CAG)15 form similar, but distinguishable hairpin structures.

The trinucleotide repeat sequence d(CGG)15 forms a heat-stable hairpin containing Gsyn. Ganti base pairs.

To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), electrophoretic mobility, chemical modification, and P1 nuclease studies were performed with a single-stranded (ss) oligonucleotide containing (CGG)15 [ss(CGG)15]. The results suggest that ss(CGG)15 forms a hairpin with the following features: (i) a stem containing Gsyn. Ganti base pairs; (ii) at > or = 200 mM K+, CGG repeats on the 5' portion of the stem base-paired to GCG repeats on the 3' side (referred to as the (b) alignment); and (iii) heat stability (Tm = 75 degrees C in low ionic strength). At < or = 100 mM K+, dimethyl sulfate reactions indicated that the hairpin in the (b) alignment was in equilibrium with another structure, presumably a hairpin in the alternative (a) alignment (CGG repeats on the 5' portion of the stem base-paired to CGG repeats on the 3' portion of the stem). Molecular dynamics simulations suggested that the loop region of the (a) alignment contained two guanines stacked on top of one another. The same guanines in the (b) alignment were base-paired in a syn-anti arrangement. We propose that the stability of the loop partially determines the stem alignment.

Gene transfer of a putative mammalian K+ channel gene from genomic and cosmid DNA.

We have successfully transfected mouse genomic DNA containing a mutant putative K+ channel gene into murine LMTK- cells. Incorporation of the mutant gene allowed primary transformants to express the mutant cell phenotype of growth at a subthreshold K+ concentration and, furthermore, caused these primary transformants to display the same characteristics of growth and unidirectional K+ flux as the mutant cell line, LTK-1, from which the genomic DNA was derived. A cosmid genomic library was formed from LTK-1 mutant DNA and shown by transfection experiments to contain at least one unique cosmid clone containing a functional copy of the mutant putative K+ channel gene.

The binding-site sizes of Escherichia coli single-stranded-DNA-binding protein and mammalian replication protein A are 65 and >/= 54 nucleotides respectively.

The electrophoretic mobilities of complexes formed with single-stranded (ss) DNA and tetrameric Escherichia coli ssDNA-binding protein (EcoSSB) or mammalian replication protein A (RPA) were analysed. The electrophoretic mobilities of the complexes in a native polyacrylamide gel increased as the lengths of the DNA increased from 28 to 70 nt, thus revealing paradoxical 'descending-staircase' patterns. Increases in the electrophoretic mobilities of EcoSSB.ssDNA complexes were observed when the lengths of the bound DNA were increased by 1 nt. Quantitative analyses of the complexes suggested that the binding-sites sizes of EcoSSB and RPA were 65 and >=54 nt respectively. The binding-site size for RPA is at least 24 nt larger than previously reported.

Anomalous cross-linking by mechlorethamine of DNA duplexes containing C-C mismatch pairs.

Nitrogen mustards such as mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures.

Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase).

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.

Hairpin properties of single-stranded DNA containing a GC-rich triplet repeat: (CTG)15.

Although triplet repeat DNA sequences are scattered throughout the human genome, their biological function remains obscure. To aid in correlating potential structures of these nucleic acids with their function, we propose their classification based on the presence or absence of a palindromic dinucleotide within the triplet, the G + C content, and the presence or absence of a homopolymer. Five classes of double-stranded (ds) triplet repeats are distinguished. Class I repeats, which are defined by the presence of a GC or CG palindrome, have the lowest base stacking energies, exhibit the lowest rates of slippage synthesis [Schlötterer and Tautz (1992) Nucleic Acids Res., 20, 211] and are uniquely associated with triplet repeat expansion diseases. The six single-stranded (ss) triplet repeats within Class I also have the potential to form hairpin structures, as determined by energy minimization. To explore the possibility of hairpin formation by ss Class I triplet repeats, studies were performed with a ss oligonucleotide containing 15 prototypic CTG repeats [ss (CTG)15]. Electrophoretic, P1 nuclease and KMnO4 oxidation data demonstrate that ss (CTG)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Potential functions of hairpins containing Class I triplet repeats are discussed with respect to protein translation and mRNA splicing. Further, potential roles of hairpin structures in triplet repeat expansion events are discussed.

Transcriptional regulation of the neuronal L-type calcium channel alpha 1D subunit gene.

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

The trinucleotide repeat sequence d(GTC)15 adopts a hairpin conformation.

The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.

At physiological pH, d(CCG)15 forms a hairpin containing protonated cytosines and a distorted helix.

To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), the structure of a single-stranded (ss) oligonucleotide containing d(CCG)15 [ss(CCG)15] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV absorbance, circular dichroism, chemical modification, and P1 nuclease digestion. ss(CCG)15 had an unusually high pKa (7.7 +/- 0.2). At pH 8.5, ss(CCG)15 formed a relatively unstable (Tm = 30 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C x +C base pairs, increased thermal stability (Tm = 37 degrees C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped away from the central portion of the helix. Examination of ss(CCG)18 and ss(CCG)20, which were designed to adopt hairpins containing alternative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pKas of approximately 8.2 and approximately 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)(n > or = 15) adopt hairpin conformations that contain CpG rather than GpC base-pair steps; the mismatched cytosines are protonated at physiological pH but are not H-bonded. We propose that protonation arises from the stacking of two cytosines in the minor groove of a distorted helix.

Prostate-Specific Ets (PSE) factor: a novel marker for detection of metastatic breast cancer in axillary lymph nodes.

Prostate Specific Ets factor is a recently identified transcriptional activator that is overexpressed in prostate cancer. To determine whether this gene is overexpressed in breast cancer, we performed a virtual Northern blot using data available online at the Cancer Genome Anatomy Project website. Ninety-five SAGE libraries were probed with a unique sequence tag to the Prostate Specific Ets gene. The results indicate that Prostate Specific Ets is expressed in 14 out of 15 breast cancer libraries (93%), nine out of 10 prostate cancer libraries (90%), three out of 40 libraries from other cancers (7.5%), and four out of 30 normal tissue libraries (13%). To determine the possibility that the Prostate Specific Ets gene is a novel marker for detection of metastatic breast cancer in axillary lymph nodes, quantitative real-time RT-PCR analyses were performed. The mean level of Prostate Specific Ets expression in lymph nodes containing metastatic breast cancer (n=22) was 410-fold higher than in normal lymph node (n=51). A receiver operator characteristic curve analysis indicated that Prostate Specific Ets was overexpressed in 18 out of 22 lymph nodes containing metastatic breast cancer (82%). The receiver operator characteristic curve analysis also indicated that the diagnostic accuracy of the Prostate Specific Ets gene for detection of metastatic breast cancer in axillary lymph nodes was 0.949. These results provide evidence that Prostate Specific Ets is a potentially informative novel marker for detection of metastatic breast cancer in axillary lymph nodes, and should be included in any study that involves molecular profiling of breast cancer.

Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel.

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10(5)-fold above normal mean expression, whereas PIP was overexpressed from 30- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.