Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

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SynopsisThe equilibrium and the stoichiometry for the reversible complexing of silver ion by DNA have been studied by potentiometric titrations, proton release pH-stat titrations, and by spectrophotometry. The complexing reactions involve primarily the purine and pyrimidine residues, not the phosphate groups. There are at lea& three types of binding (types I, 11, and 111), of which the h t two have been intensively studied in this work. The sum of type I and type I1 binding saturates at one silver atom per two nucleotide residues. In the type I and type I1 reactions, zero and one proton, reapectively, are displaced per silver ion bound. At p H 5.6, the reactions occur stepwise, type I being first, while at pH 8.0, they occur simultaneously. The silver ion binding curve is very sharp at pH 8, indicating a cooperative reaction. The strength of the binding increases with increasing GC content. Type I binding is more important for GCrich DNA's than for GC-poor ones. Denatured DNA binds more strongly than does native DNA. The silver ion complexing reaction is chemically and biologically reversible. We propose that type I1 binding essentially involves the conversion of an N-H . . . N hydrogen bond of a complementary base pair to an N-Ag-N bond. The nature of type I binding is less clear, but it may involve a T interaction with stacked bases. The buoyant density ( p a ) of DNA in a Cs2S04 density gradient increases when the DNA reacts with silver ion. The buoyant density change is about 0.15 g./ml. for 0.5 silver bound per nucleotide. The large buoyant density changes and the selective nature of the complexing reaction make it possible to perform good separations between native and denatured DNA or between GC-rich and GC-poor native DNA's by density gradient centrifugation.

Due to problems with primary tumor cell culture, conventional cytogenetics has yielded little insightful information on chromosomal alterations in prostate cancer. The primary aim of this study was to define the ability of comparative genomic hybridization (CGH) to detect and map genetic deletions in prostate tumors. A secondary aim was to apply multiple assays to individual tumors as a means of deciphering the mechanisms of genetic alterations in prostate cancer. CGH results were compared with allelic imbalance measurements at 29 distinct loci on chromosome 8 in 18 specimens (17 malignant and 1 benign). CGH detected no changes in cases where all informative PCR/RFLP loci were retained and detected all p arm deletions consisting of at least two loci. We estimate that in this study, the smallest deletions detected by CGH were approximately 20-30 cM. Physical mapping of subchromosomal arm deletions by CGH correlated well with allelic imbalance mapping by PCR/RFLP: The data agreed at 88% of loci on 8p and 92% of loci on 8q. Fluorescence in situ hybridization (FISH) with multiple centromere probes and DNA content flow cytometry (FCM) also was performed on selected specimens. FISH revealed two cases of chromosome 8 aneusomy. In these two cases and three others, CGH showed simultaneous p arm deletion and q arm gain, suggesting isochromosome 8q formation. Together, these data suggested that, simple chromosomal aberrations were responsible for allelic losses on 8p and allelic gains on 8q in a significant number of prostate tumors. We also used CGH to examine relative DNA sequence copy number throughout the genome. Changes frequently associated with 8p loss include gains of 8q and losses of 13q, 16p, 16q, 17p, 17q, 20q, and Y. Cases with 8p loss exhibited five times the number of alterations as did cases without 8p loss.

The glycophorin A assay was used to estimate the frequency of mutations that accumulate in vivo in somatic cells of persons with Bloom's syndrome (BS). This assay measures the frequency in persons of blood type MN of variant erythrocytes that lack the expression of one allelic form of glycophorin A, presumably due to mutational or recombinational events in erythroid precursor cells. Samples of blood from persons with BS showed dramatic 50-to 100-fold increases in the frequency of variants of three types, those with a hemizygous phenotype, those with a homozygous phenotype, and those with what appears to be partial loss of the expression of one locus. The high frequency of homozygous variants, genetic evidence for altered allelic segregation of a specific biochemical locus, provides evidence for increased somatic crossing-over in vivo in BS. An increased generation of functional hemizygosity and homozygosity in their somatic cells may play an important role in the extreme cancer risk of persons with BS.Bloom's syndrome (BS) (1) is a rare autosomal recessive genetic disorder of growth that greatly increases the affected individual's chance of developing cancer. In the 130 affected individuals in the Bloom's Syndrome Registry, 57 malignant neoplasms have been detected, at a mean age at diagnosis of 24.7 years (2). A wide spectrum of tumor types has been observed in affected individuals, including lymphoid and myeloid leukemias, lymphomas, squamous cell carcinomas, and adenocarcinomas (3).In addition, a distinctive array of cytological abnormalities in a variety of cell types, signifying a remarkable degree of genomic instability, is a constant feature of BS (4, 5). These include high spontaneous frequencies of chromosome breaks and rearrangements (5-8), sister-chromatid exchanges (SCEs) (5, 9-11), micronucleated cells (12, 13), and cells with specific-locus mutations (14-17). A unique cellular abnormality in untreated BS cells in vitro is a dramatic increase over normal in the frequency of a certain type of quadriradial configuration (Qr) in metaphase cells (5,18). The Qrs of BS are the microscopically visible consequence of interchanges that, earlier in the cell cycle, occurred between homologous chromosomes at apparently homologous sites; they are interpreted as cytological evidence for an increased frequency of somatic crossing-over in BS (18)(19)(20)(21). Heterozygous carriers of the BS mutation (bl/+) are clinically and cytologically normal (22). Although the primary genetic defect responsible for BS may not have been defined, DNA ligase I activity has been reported to be decreased (23)(24)(25), and this very possibly is responsible for the genomic instability of BS. The complex cytogenetic abnormalities readily demonstrable in BS cells in vitro raise the possibility that somatic cell mutation (point mutation, chromosome segmental rearrangement, and somatic recombination) is also substantially increased in vivo in BS, and this could explain the extreme cancer risk.In the present study we have used the ...

An assay method is described for determining the frequency of human erythrocytes having a gene expression loss phenotype at the glycophorin A locus presumably due to in vivo somatic mutational events in erythroid precursor cells. Monoclonal antibodies specific for the M and N glycophorin A alleles are used to identify variant cells that lack the expression of one allele in blood samples from MN heterozygotes. Flow cytometry and sorting are used to enumerate and purify variant cells. Using three different antibody combinations which are sensitive to the loss of either the M or the N allele, we find that variant cells occur at a frequency of 1 X 10(-5) in normal donors. We also detect variant cells with an apparent homozygous phenotype suggesting that events leading to homozygosity may occur at similar frequencies to gene loss events. Significant increases in variant cell frequency are observed in cancer patients after exposure to mutagenic chemotherapy drugs.

An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described l W 1 assay that required a dual-beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1 W l assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.

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