Ronald H. Jensen

Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction

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.

Measurements of the frequency of human erythrocytes with gene expression loss phenotypes at the glycophorin A locus

SummaryAn 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 1x10-5 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.

Monoclonal antibodies specific for the M- and N-forms of human glycophorin A.

Four mouse monoclonal antibodies directed against the red cell membrane protein glycophorin A have been isolated and characterized. They are produced by hybridomas derived from SP2/0 myeloma cells and spleen cells from Biozzi mice immunized with a mixture of human erythrocytes from homozygous blood group M and N individuals. These antibodies recognize and bind to purified glycophorin A and to glycophorin on the red cell surface. All are of the IgGl, kappa light chain subclass and bind to determinants presented on the 39 amino acid, trypsin-sensitive, N-terminal peptide of glycophorin A. Three display differential specificities for the two allelic forms of glycophorin A; two are exquisitely specific for the M-form and one preferentially binds the N-form. Treatment of red cells with neuraminidase, which removes N-acetylneuraminic acid from glycophorin A, abolishes the binding of these three antibodies. The binding of the N-specific antibody is also sensitive to modification of the amino-terminal residue of the antigen. The fourth antibody binds equally well to both the M- and N-forms as well as to neuraminidase-treated red cells; thus it recognizes a public, N-acetylneuraminic acid independent glycophorin A determinant.

Biological dosimetry of radiation workers at the Sellafield nuclear facility.

The British Nuclear Fuels plc facility at Sellafield performs a range of nuclear-related activities. The site has been in operation since 1950 and has, in general, employed a stable work force, many of whom have accumulated relatively high occupational exposures to ionizing radiation. This paper compares the physical dosimetry with two biological end points for evaluating radiation exposure: fluorescence in situ hybridization with whole-chromosome painting probes to quantify stable chromosome aberrations (translocations and insertions), and glycophorin A (GPA) analysis of variant erythrocytes. For the cytogenetic analyses, 81 workers were evaluated in five dose categories, including 23 with minimal radiation exposure (< or = 50 mSv) and 58 with exposures ranging from 173 to 1108 mSv, all but 3 being > 500 mSv. In a univariate analysis, the mean stable chromosome aberration frequencies showed a significant increase with dose category (P = 0.032), and with cumulative dose when dose is treated as a continuous variable (P = 0.015). The slope of the dose response for stable aberrations is 0.79 +/- 0.22 aberrations per 100 cells per sievert (adjusted for smoking status), which is less than that observed among atomic bomb survivors, and suggests a dose and dose-rate effectiveness factor for chronic exposure of about 6. Analyses of the data for GPA N/O and N/N variants from 36 workers revealed no correlation with dose. Neither was there a correlation between the frequencies of N/O GPA variants and stable aberrations, although a weak negative association was observed between N/N variant frequency and stable aberrations (r = -0.38, P = 0.05). These results provide clear evidence for the accumulation of stable aberrations under conditions of chronic occupational exposure to ionizing radiation and show that stable chromosome aberrations are a more sensitive indicator for chronic radiation exposure than GPA variants. In comparison with human studies of brief exposure, chronic low-dose exposures appear substantially less effective for producing somatic effects as reflected by stable chromosome aberrations.

MAPPING OF REGIONS OF PHYSICAL DELETION ON CHROMOSOME 16q IN PROSTATE CANCER CELLS BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Recent evidence suggests that a tumor suppressor gene important in the progression of prostatic carcinoma may reside on chromosome 16q. The exact location and identity of this gene are unknown. We used fluorescence in situ hybridization in a novel manner to define more clearly the location of this gene. Region-specific chromosome 16 cosmid contig probes were hybridized directly to interphase prostatic carcinoma nuclei in order to measure physical deletion of chromosomal loci. Fifteen of 30 tumors (50%) showed evidence of physical deletion of 16q24. Other probes were used to test for regions of chromosome 16 deletions in the same specimens, and a map showing a region of common deletion was created. This map showed the proximal terminus of the region of common deletion to be located distal to 16q23.1. These data correlate well with loss of heterozygosity data in the literature and provide further evidence for the presence of a prostatic carcinoma tumor suppressor gene on chromosome 16q.

The effect of chemotherapy on the in vivo frequency of glycophorin A ‘null’ variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.

A study of the effects of exposure on cleanup workers at the Chernobyl nuclear reactor accident using multiple end points

Blood samples were collected from 192 exposed workers who participated in the cleanup after the April 26, 1986, nuclear reactor accident at Chernobyl, Ukraine. These samples, together with samples from 73 individuals living in Russia but not involved in Chernobyl cleanup activities, were collected during September 1991 to May 1996 and shipped to the U.S. for evaluation by three bioassays: cytogenetic analysis based on chromosome painting, HPRT mutation analysis and glycophorin A (GPA) variant analysis. Univariate statistical analyses of the results of each bioassay (including adjustments for age, smoking status and estimated precision of the bioassay) found greater frequencies of chromosome translocations and HPRT mutant T lymphocytes among the exposed individuals compared to the controls (P < or = 0.01). GPA analyses showed no significant difference for exposed compared to controls for either hemizygous, N/O, or homozygous, N/N, variant cell frequency. Multivariate analysis of variance of the subset of 44 exposed and 14 unexposed individuals with measurements from all three bioassays found elevated frequencies of chromosomal translocations and HPRT mutants, and reduced frequencies for both GPA end points among the exposed persons compared to the controls. However, none of these differences, considered singly or in combination, was statistically significant (although statistical power is low due to small sample sizes). Mean estimated dose, based on cytogenetic response, for those exposed was 9 cGy (range 0 to 51 cGy) and was less than that estimated by physical dosimetry (25 cGy). Correlation between the end points of the bioassays and estimated physical dosimetry was low (r < 0.2); the only significant correlation found was for physical dose estimate and dates worked at Chernobyl (r = 0.4, P < 0.01), with those working soon after the accident receiving greater estimated doses.

Elevated Frequency of Glycophorin A Mutations in Erythrocytes from Chernobyl Accident Victims

In 1986, when an explosion accident occurred at the Chernobyl, Ukraine nuclear power plant, a large number of people were exposed to significant amounts of ionizing radiation. During the time between 1986 and 1992, peripheral blood samples were obtained from 102 people who either were on site during the emergency or were brought to Chernobyl shortly thereafter to assist in the cleanup of radioactive contaminants and isolate the damaged reactor from the environment. These blood samples plus samples from 13 unexposed Soviet individuals were analyzed by flow cytometry using the allele-loss somatic mutation assay for glycophorin A. Results of these assays show that the frequency of N/O variant red cells increased in proportion to the estimated radiation exposure of each individual. The radiation dose-response function derived from this population closely resembles that determined previously for atomic bomb survivors whose blood samples were obtained and analyzed 40 years after their exposure. This suggests comparable mutation induction per unit dose for these two populations and long-term persistence of the mutational damage. In addition, measurements on multiple blood samples from each of 10 donors taken over a 7-year period showed no significant changes in N/O variant cell frequencies, confirming the persistence of radiation-induced somatic mutations in long-lived bone marrow stem cells.

Interactions Between Pairs of DNA-Specific Fluorescent Stains Bound to Mammalian Cells'

The interactions between DNA-specific fluorescence stains complexed with mitotic Chinese hamster cells were studied by spectrofluorometric and flow fluorometric techniques. The degree of binding interactions and of energy transfer between stains was determined from the intensities and shapes of fluorescence emission spectra of cells complexed with pairs of stains. The stain pairs Hoechst 33258-chromomycin A3, Hoechst 33258-ethidium bromide, and chromomycin A3-ethidium bromide exhibited efficient energy transfer from the short wavelength absorber (donor) to the long wavelength absorber (acceptor), and little competitive or cooperative binding of stains. The stain pair quinacrine-ethidium bromide exhibited both energy transfer and competitive binding. None of the stain pairs showed evidence of strong electronic interactions between stains. The magnitude of energy transfer interactions was used to estimate the quantity and distribution of the stains molecules complexed to mitotic cells. The results indicate a fairly even distribution of each of these stains along the DNA of intracellular mitotic chromosomes.

Biodosimetry of Chernobyl cleanup workers from estonia and latvia using the glycophorin A in vivo somatic cell mutation assay

The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method.