A modification of the Hedley‐method for flow cytometric DNA analysis of paraffin‐embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core‐biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made it easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S‐phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high‐polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus.

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An easy method for preparation of bare cell nuclei from fresh solid tissues for DNA flow cytometry is described. Pieces of up to 2 x 2 x 2 mm3 size from fresh tissues were fixed in formalin. After removal of formalin by washing with ethanol and rehydration with tap water, the tissue pieces were incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly with DAPI. Staining with ethidium bromide gave unsatisfactory results. Neither mechanical disaggregation nor centrifugation were used. The resulting cell nucleus suspensions had extremely low frequencies of debris particles and of clumped cell nuclei. A good yield, a minimized loss, and a good stainability of cell nuclei were obtained.The applicability of the method was exemplified by the analysis of biopsies from the colon-rectum in patients with ulcerative colitis and of biopsies from the bladder in patients with bladder cancer and compared to the standard method of this laboratory, which uses mechanical disaggregation, ethanol fixation, pepsin treatment, and staining with ethidium bromide. The formalin-subtilisin Carlsberg technique resulted in good agreement of ploidy measurements compared to the standard method, a higher number of evaluable histograms, an improved detectability of aneuploid cell populations, and an improved accuracy of the S-and G2-phase analysis, particularly in samples with low proliferation. The method also makes it possible to use long-term storage and to transport samples by post. 0 1993 Wiley-Liss, Inc.Key terms: DNA flow cytometry, solid tissues, formalin fixation, subtilisin Carlsberg DNA flow cytometry of solid tissues is a well-established technique for the characterization of tumor ploidy and proliferation. Several methods for the dissociation of fresh, solid tissues into single-cell or nuclear suspensions are in use and there are two main groups of techniques: enzymatic and detergent-based procedures, both of which also use mechanical treatment.A previously published improved Hedley-method for the preparation of paraffin-embedded tissues for flow cytometric DNA analysis has proved useful for the examination of various types of tumor tissues (8). In this preparation, the dewaxed and rehydrated tissue, which had been fixed with formalin, is treated with subtilisin Carlsberg. Since all centrifugation steps are omitted, the loss of material is minimized and the suspensions show extremely low frequencies of clumped cell nuclei and clumped debris particles. In this way the analysis of ploidy and S-phase in archival material can be considerably improved.The good results obtained with this method were an encouragement for us to use its essential parts also for the preparation of fresh biopsies; it was a n intent to achieve, in such preparations, both low portions of clumpings and low frequencies of debris particles. It had been observed that subtilisin Carlsberg treatment of formalin fixed tissue yields suspensions of stable nuclei which remain intact even after prolonged incubation with the enzyme (8) an...

Background. Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. Material and methods. Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 Böcking’s grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow‐ and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. Results. By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. Conclusion. Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.

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Both systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are autoimmune diseases sharing similar genetic backgrounds. Genome-wide association studies have constantly disclosed numerous genetic variants conferring to both disease risks at 7q32.1, but the functional mechanisms underlying them are still largely unknown. Through a series of bioinformatics and functional analyses, we prioritized a potential independent functional single-nucleotide polymorphism (rs13239597) within TNPO3 promoter region, residing in a putative enhancer element and validated that IRF5 is the distal target gene (w118 kb) of rs13239597, which is a key regulator involved in pathogenic autoantibody dysregulation, increasing risk of both SLE and SSc. We experimentally validated the long-range chromatin interactions between rs13239597 and IRF5 using chromosome conformation capture assay. We further demonstrated that rs13239597-A acted as an allele-specific enhancer regulating IRF5 expression, independently of TNPO3 by using dual-luciferase reporter assays and CRISPR-Cas9. Particularly, the transcription factor EVI1 could preferentially bind to rs13239597-A allele and increase the enhancer activity to regulate IRF5 expression. Taken together, our results uncovered a mechanistic insight of a noncoding functional variant acting as an allele-specific distal enhancer to directly modulate IRF5 expression, which might obligate in understanding of complex genetic architectures of SLE and SSc pathogenesis.

RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.

The cytosolic thymidine kinase 1 (TK1) is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti‐TK1 antibody works in archival specimens and is a specific marker of cell proliferation.